Enabling cholesterol catabolism in human cells

ABSTRACT

Compositions, methods, and systems for modifying sterol metabolism in a subject is disclosed. In some embodiments, the subjects may be administered one or more mammalian cells modified to express at least one sterol degrading enzyme derived from a bacterium. In many embodiments, the cell is a macrophage or monocyte stably expressing three or more enzymes that aid in opening the β ring of cholesterol. The disclosed compositions and methods may be useful in lowering cholesterol levels in a subject in need thereof. In some embodiments, the subject may have a genetic predisposition to atherosclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority pursuant to 35 U.S.C. §119(e) of U.S. provisional patent application No. 62/754,499 entitled“ENABLING CHOLESTEROL CATABOLISM IN HUMAN CELLS,” filed on 1 Nov. 2019,which is hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under grant numberHL110937 awarded by the National Institutes of Health of the U.S.Department of Health and Human Services. The government has certainrights in the invention.

FIELD

The processes, methods, compositions, and systems disclosed herein areuseful in regulating sterol concentrations in subjects in need thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 29, 2019, isnamed P278091_US_03_507078-00005_SL.txt and is 105,499 bytes in size.

BACKGROUND

Atherosclerosis is a chronic maladaptive inflammatory response initiatedby the retention of cholesterol rich apolipoprotein B-containinglipoproteins within the arterial wall. Atherosclerosis is an underlyingcause of cardiovascular disease (CVD), myocardial infarction, stroke andperipheral vascular disease, which are leading causes of death in theUnited States. CVD originates from aberrations in normal lipidmetabolism (some lifestyle choices, some genetic) that result inelevated plasma lipoproteins (principally LDLs) and/or low levels ofhigh-density lipoproteins (HDLs). CVD is often an age dependent,progressive disease that is largely undetected or ignored until an event(i.e. myocardial infarction or stroke) occurs in the later stages ofdisease. Therefore, current therapies focus on preventing a second event(or a primary event in high risk individuals) by reducing thecirculating levels of LDLs and/or increasing HDLs.

SUMMARY

The present disclosure is directed to therapies targeting thebiochemical basis of CVD (at a biochemical level the inability ofmacrophages to modify cholesterol to degrade the cholestane ring ofcholesterol is a fundamental component of CVD). Applicants hypothesizedthat if macrophages had the ability to degrade cholesterol, they mightnot become engorged with cholesterol/cholesterol esters and elicit themaladaptive immune response that leads to the onset and progression ofatherosclerosis. The present compositions and methods are based, inpart, on Applicant's surprising observation that during chronicinfection Mycobacteria tuberculosis survival in humans is enabled bytheir ability to feed on cholesterol, while contained within foamymacrophages.

Disclosed herein are methods to humanize and express the enzymes thataid in catalyzing cholesterol degradation, including side chainmodification, ring modifications, and modifications leading to ringopening. Disclosed herein are methods for enzyme-mediated cholestanering opening in human cells. The present disclosure will aid thedevelopment of genetic and cell-based therapies allowing for an entirelynew and inventive approach for the medical management of CVD.

Disclosed herein are methods, compositions, and systems for regulatingsterol metabolism. In one embodiment, the disclosed compositions,methods, and systems may enable sterol catabolism in a mammal. In someembodiments, the disclosed methods, compositions, and systems may beuseful in modifying mammalian cells to express one or more non-mammalianenzymes active in sterol catabolism. In some embodiments the mammaliancells may be immune cells, such as monocytes. In some embodiments themonocytes are macrophages. In some embodiments, the sterol may becholesterol.

Disclosed herein are methods for modifying a mammalian cell with nucleicacid compositions that enable and/or promote expression of one or moreproteins useful in degrading a sterol. In some embodiments, thecomposition includes a vector having one or more control sequences forpromoting the expression of one or more protein coding sequences. Insome embodiments, the vector is a viral vector or a transposableelement.

Disclosed herein are methods of expressing proteins in a cell that doesnot normally express such proteins. In many embodiments the proteins maybe enzymes capable of altering a sterol, such as cholesterol orderivatives thereof. In some embodiments, the enzymes are selected fromcholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase(Δ1-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), andP450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx). Insome embodiments, the enzymes may be derived from one or morenon-eukaryotic organisms, for example bacteria. In most embodiments, theamino acid sequences of and/or the coding sequences for these enzymeshas been modified to aid in expressing the enzymes in a eukaryotic cell.

Disclosed herein are methods and compositions useful in creatingmodified human cells capable of degrading cholesterol. In manyembodiments, the human cells are immune cells comprising one or morenucleic acid sequences coding for one or more proteins useful indegrading a sterol, or derivative thereof. In many embodiments, theproteins may be derived from a non-eukaryote, such as bacteria. In manyembodiments, the cells are immune cells, for example monocytes or, moreparticularly, macrophages. In some embodiments, the macrophages may bemodified to degrade low density lipoproteins associated withatherosclerotic plaques.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Development of an atherosclerotic plaque. Upon endothelialdysfunction or damage, atherosclerotic lesions begin to develop.Interruption of the endothelial barrier allows for the infiltration andaccumulation of LDLs in the intima, initiating an immune response forthe recruitment of monocytes. Upon entering the subendothelialspace,monocytes differentiate into macrophages, which engulf thecholesterol/cholesterol ester (CE)-rich lipoproteins via LDL- andscavenger receptor mediated endocytosis. LDL-cholesterol accumulation inmacrophages that infiltrate the intima initiates a chronic inflammatoryresponse, resulting in the recruitment of more macrophages. With timethe macrophages become engorged with CE and transform into foam cells.This maladaptive immune response leads to the accumulation of foamscells within the intima resulting in the formation of cholesterolplaques.

FIG. 2A. Simplified summary of human cholesterol synthesis andmetabolism. All carbons of cholesterol are derived from acetyl-CoA. Tosynthesize cholesterol, acetyl-CoA is converted to a five carbonintermediate known as an isoprene unit. Six isoprene units are condensedto form squalene, the 30 carbon linear precursor of cholesterol.Squalene is cyclized by squalene synthase to produce lanosterol, formingthe tetracyclic steroid skeleton (cholestane ring). Following anadditional nineteen enzymatic steps, lanosterol is converted tocholesterol. Cholesterol is used in the production of bile salts andsteroid hormones. However, one key feature of cholesterol metabolism isthat once squalene is cyclized and the cholestane ring is formed, thering cannot be opened enzymaticallyin human cells.

FIG. 2B. Cholesterol catabolism in Mycobacteria. The aliphatic sidechain (C17) is removed in a process similar to beta-oxidation (sidechain degradation pathway), and ring-opening is mediated by thefour-ring degradation pathway. In ring degradation, ADD,4-androstenedione is acted upon by two enzymes,3-ketosteroid-Δ1-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase(KshA/B), which catalyze B-ring opening and aromatization of ring A toproduce 3-HSA (3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione).Brackets designate an intermediate compound that degrades spontaneously.This Example was adapted from Vander Geize et al.

FIG. 2C Shows HPLC-analysis of cholesterol catabolism. Humanized hCD andhACMB expressed in E. coli were partially purified. [¹⁴C]-spikedcholesterol (100 nCi/10 μM) was then added to a mixture containing hCDand hAMCB. After 24 hours the samples were extracted with ethylacetateand catabolites were identified by their retention time when resolvedusing RP-HPLC (C-18 column) in combination with the spectral shift thatis produced (ie. cholesterol does not absorb at 245 nm and elutes with aretention time of 40 minutes. Cholestenone and CDD absorb strongly at˜245 nm and have shorter retention times. Control E. coli extractsdemonstrated no metabolic activity against cholesterol, CDD, or any ofthe catabolites produced prior to ring opening, which makes detection ofsterol derived analytes robust.

FIG. 2D shows Co-crystal structure of bacterial KstD in complex with ADD(left). Model of cholesterol in the KstD active site (right). Isoleucineresidues producing a steric clash with the C17-side chain are shown inyellow. Coordinates for Pymol generated figures and for modeling werekindly provided by Ali. Rohman et al.

FIG. 2E Shows how the combined actions of hKstD and hKshA/B open thecholestane ring. HPLC profiles documenting the conversion of[¹⁴C]-labeled PD to 9-OHPD, PDD, and 3-HSP by the actions of hKshA/B,hKstD, or a mixture of hKshA/B and hKstD, respectively. 10 μM PD spikedwith 100 nCi [¹⁴C]-PD was mixed with the partially purified enzymesindicated. After 24 hours the samples were extracted and analyzed byRP-HPLC above. A second spectral shift is produced with B-ring openingand aromatization of ring A (i.e. conversion of PDD or 9-OHPD to 3-HSP).PD, 9-OHPD, and PDD all have and an absorbance maximum of ˜245 nM; 3-HSPabsorbance maximum is 280 nm; lower left corner). This data is clean androbust, because control E. coli extracts do not readily catabolize ormetabolize any of the compounds that retain the cholestane ring. MS datarevealed an identical mass match for 3-HSP.

FIG. 2F is a representative HPLC elution profile showing PD is onlyconverted to PDD by Hep3B cells that have been transfected with hKstD(Panel B).

FIG. 2G shows that KshA/B (3-ketosteroid-9α-hydroxylase) is a twocomponent oxgenase that utilizes molecular oxygen and NADH as acofactor. The crystal structure shows that three KshA-subunits areassembled “head to tail” with electrons transferred for the iron sulfurcomplex in the “head” of one protein to the Fe2+ contained in the “tail”of another. Image derived from PDB ID 2ZYL.

FIG. 2H is a diagram of the “2A-peptide” bicistronic expression. Anexpression construct was produced in which hKshA and hKshB are encodedto produce a single in-frame mRNA with the 2A peptide from Porcineteshovirus-1 separating hKshA from hKshB. To help determine theefficiency of 2A-mediated release, FLAG and HA recognition sequenceswere added to hKshA and KshB, as illustrated. Human Hep3B cells weretransfected with the construct, and levels of hKshA and hKshB weredetected by western analysis using the anti-FLAG and anti-HA antibodiesat 24, 48 and 72 hours after transfection. Lane C represents lysatesfrom Hep3B transfected with PP5-FLAG as a control for detection byanti-FLAG antibody. The levels of KshA and KshB are similar. If readthrough of the 2A peptide had occurred, a band of ˜90 kD should havebeen detected by both anti-FLAG and anti-HA antibodies. Based on thelack of a 90 kD band the release of the KshA appears to be extremelyefficient.

FIG. 2I shows RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from progesterone (PD) by Hep3B cells transiently expressingMTS-KshAB and Δ¹-KstD. Representative 2-D chromatograms at (Panel b) I245 nm, (Panel b)(Panel b) I 280 nm, and (Panel c)(Panel c) C4-¹⁴Cscintillation events from Hep3B cells transiently expressing EF1α drivenMTS-KshAB and Δ¹-KstD. Cells were incubated with 15.7 mg (10 mM)progesterone spiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) andtime points taken at 6, 12, 24, and 36 hours. Analysis at (Panel b) I245 nm reveals a large proportion of the PD substrate being converted to9-hydroxypregn-4-ene-3,20,dione (9-OHPD, t_(r)=5.2 min) by 6 hours.Although pregn-1,4-diene-3,20-dione (PDD; t_(r)=10.0 min) is notobserved at the 6 hour time point, analysis of (Panel b)(Panel b) I 280nm and (Panel c)(Panel c) C4-¹⁴C scintillation events reveals theformation of 3-HSP (t_(r)=7.2 min, I_(max) 280 nm). By 12 hours, the PDsubstrate and 9-OHPD product are exhausted resulting in maximalproduction of 3-HSP. Interestingly, both the area and counts under thecurve of 3-HSP decreases at further time points, suggesting that Hep3Bcells have ability to further modify the pregnane ring once opened.Evidence of this can be observed at 24 and 36 hour time points as newC4-¹⁴C scintillation events appear between 6.0-6.5 minutes.

FIG. 2J is a western blot analysis of Hep3B cells expressing EF1α drivenMTS-KshAB-P2A-Δ¹-KstD or MTS-KshAB-T2A-Δ¹-KstD constructs. Hep3B cellswere transiently transfected with pDest51-KshAB-P2A-Δ¹-KstD orpDest51-KshAB-T2A-Δ¹-KstD plasmids in 60 mm dishes and proteinexpression was assessed following 48 hours incubation. Cells werecollected by scraping in 500 mL RIPA buffer and mechanically lysed onice using a syringe with a 27 gauge needle. Protein samples were mixedwith an equal volume of 2× Laemmli sample buffer, boiled for 5 min, andspun at 15,000×g for 10 min at 4° C. Protein samples (25 mg) wereseparated using SDS-PAGE on a 10% polyacrylamide gel, transferred toPVDF membranes, and probed with anti-FLAG (1:1000) or anti-HA (1:3000).ECL anti-mouse IgG secondary antibody conjugated to HRP (1:10,000) andSuperSignal West Femto Substrate was used for detection. Samples includethe P2A construct, T2A construct, Hep3B CMV-MTS KshAB cell line(positive KshA FLAG and KshB HA control), Hep3B CMV-Δ¹-KstD cell line(positive Δ¹-KstD FLAG control), and non-transduced Hep3B cells(negative control).

FIG. 2K shows an RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from progesterone (PD) catabolism by Hep3B cells expressingEF1α driven KshAB P2A Δ¹-KstD or KshAB T2A Δ¹-KstD constructs. Hep3Bcells were transiently transfected with pDest51-KshAB P2A Δ¹-KstD orpDest51-KshAB T2A Δ¹-KstD plasmids in 60 mm dishes. Following 48 hoursof protein expression, cells were incubated with 15.7 mg (10 mM) PDspiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) for 48 hours.Representative 2-D chromatograms at (Panels a & d) I 245 nm, (Panels b &e) I 280 nm, and (Panels c & f) C4-¹⁴C scintillation events demonstratethe efficiency of the P2A and T2A constructs in producing 3-HSP(t_(r)=7.2 min, I_(max) 280 nm) through PD catabolism. Analysis of theP2A construct at (Panel b) I 245 nm reveals a large proportion of the PDsubstrate being converted to 9-hydroxypregn-4-ene-3,20,dione (9-OHPD,t_(r)=5.2 min) by 48 hours. However, in comparison to the T2A constructat (Panel d) I 245 nm, residual 9-OHPD is observed, suggesting Δ¹-KstDis the rate limiting step in 3-HSP (t_(r)=7.2 min, I_(max) 280 nm)formation. Although 3-HSP is not observed at (Panel b)(Panel b) I 280 nmor in (Panel c)(Panel c) C4-¹⁴C scintillation events, the accumulationof scintillation events from an unidentified metabolite (t_(r)=2.3 min)are detected prior to the 5.2 minute retention time of 9-OHPD. Incontrast, the T2A construct at (Panel d) I 245 nm reveals completereduction in the PD substrate, 9-OHPD, and pregn-1,4-diene-3,20-dione(PDD, t_(r)=10.0 min, I_(max) 247 nm) by 48 hours. In addition, (Panele) I 280 nm reveals the formation of 3-HSP. Furthermore, (Panel f)C4-¹⁴C scintillation events confirm the formation of 3-HSP as well asadditional scintillation events from unidentified metabolites prior to3-HSP's 7.2 minute retention time.

FIG. 3. Summary of lipoprotein metabolism. Lipids obtained from the dietor synthesized by the liver are packaged into and transported bylipoproteins. Dietary fats absorbed by the intestinal epithelial cellsare packaged into chylomicrons which deliver fatty acids (released fromTGs by lipoprotein lipase) to adipose tissue and muscle. The remnantsare removed from the circulation by the liver and the contents areeither utilized or repackaged into VLDLs for additional transport. AsTGs are removed from VLDLs and IDLs, they become LDLs which are enrichedwith cholesterol esters. When elevated, LDLs contribute tocardiovascular disease. Thus, current treatment options work to lowerserum LDL by targeting metabolic pathways leading to increasedexpression of LDL receptors that clear LDLS.

FIG. 4. Amelioration of atherosclerosis with cholesterol degradingmonocyte therapy. Cholesterol degrading monocytes will be redeliveredintravenously to the patient where they migrate to sites of plaqueformation within the arterial intima. Once the monocytes transmigrate tothe intima, they differentiate into macrophages. Macrophages will haveaccessibility to high levels of LDL-C which will initiate the expressionof cholesterol catabolizing enzymes leading to enzymatic degradation ofcholesterol.

FIG. 5. Cholesterol degradation pathway utilized by M. tuberculosis. M.tuberculosis and many other microorganisms (e.g. R. rhodochrous, R.erthropolis, S. denitrificans, etc.), express enzymes that enable thebacteria to degrade cholesterol. Following an independent side chainremoval pathway, enzymes sequentially modify the steroid nucleus ofcholesterol resulting in B-ring cleavage. Once the ring has been opened,cholesterol is further catabolized to acetyl-CoA, which is used formetabolic processes including lipid synthesis and the TCA cycle for theproduction of energy.

FIG. 6. Catalytic mechanism for opening the cholestane ring ofcholesterol without prior side chain removal. If the cholesteroldegrading enzymes do not require side chain removal, only three enzymesmay be necessary to open the cholestane ring. The first enzyme,cholesterol dehydrogenase (CholD) (1), is responsible for the oxidationand isomerization of Δ₅-3β-hydroxsteroids to Δ₄-ketosteroids(cholesterol (CL) to cholestenone (CN)). The presence of the 3-ketoneand isomerization of the double bond between C5 and C6 atoms of ring Bto C4 and C5 atoms of ring A are required by the last two enzymes priorto catalyzing their respective reactions. The second enzyme, anoxiccholesterol metabolism B enzyme (acmB) (2) eliminates the 1α and 2βhydrogen atoms thereby introducing a double bond between the C1 and C2atoms of ring A (cholestenone (CN) to choleste-1,4-diene-3-one (CDN)).The last enzyme, 3-ketosteroid 9α-hydroxylase (KshAB) (3), catalyzes theaddition of a hydroxyl group on C9 of ring-B (cholestenone (CN) to9-hydroxycholeste-4-ene-3-one (9-OHCN)). Both Δ₁-KstD and KshAB are ableto catalyze their respective reactions before or after the other andtheir combined activities lead to the formation of the unstableintermediate 9-hydroxycholeste-1,4-diene-3-one (9-OHCDN). Altogether,the presence of the 3-keto group, the isomerization of the double bondbetween C4 and C5, the trans-axial elimination of C1 and C2 of ring A,and the hydroxylation at C9 of ring B results in the destabilization andspontaneous opening of the cholestane ring to form the product,3-hydroxy-9,10-secocholeste-1,3,5(10)-triene-9-one (3-HSC).

FIG. 7. Catalytic mechanism for opening the cholestane ring ofcholesterol following side chain removal. Four enzymes are necessary toopen the cholestane ring. The first enzyme, P450-FdxR-Fdx (1) is afusion protein consisting of human cytochrome P450 (CYP11A1),ferrodoxinreductase, and ferrodoxin. The P450-FdxR-Fdx fusion proteinremoves the hydrophobic side chain of cholesterol (CL) to producepregnenolone (PL). Side chain removal is a critical step required by theremaining enzymes prior to catalyzing their respective reactions. Thesecond enzyme, 3β-hydroxysteroid dehydrogenase (HSD2) (2), isresponsible for the oxidation and isomerization of Δ⁵-3β-hydroxsteroidsto Δ⁴-ketosteroids (pregnenolone (PL) to progesterone (PD)). Thepresence of the 3-ketone and isomerization of the double bond between C5and C6 atoms of ring B to C4 and C5 atoms of ring A are required by thelast two enzymes prior to catalyzing their respective reactions. Thethird enzyme, 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) (3) eliminatesthe 1α and 2β hydrogen atoms thereby introducing a double bond betweenthe C1 and C2 atoms of ring A (progesterone (PD) topregn-1,4-diene-3,20-dione (PDD)). The last enzyme, 3-ketosteroid9α-hydroxylase (KshAB) (4), catalyzes the addition of a hydroxyl groupon the ring-B C9 of 3-ketosteroids (progesterone (PD) to9-hydroxypregn-4-ene-3,20-dione (9-OHPD)). Both Δ¹-KstD and KshAB areable to catalyze their respective reactions before or after the other,and their combined activities lead to the formation of the unstableintermediate 9-hydroxypregn-1,4-diene-3,20-dione (9-OHPDD). The presenceof the 3-keto group, the isomerization of the double bond between C4 andC5, the trans-axial elimination of C1 and C2 of ring A, and thehydroxylation at C9 of ring B results in the destabilization andspontaneous opening of the cholestane ring to form the product,3-hydroxy-9,10-secopregnan-1,3,5(10)-triene-9,20-dione (3-HSP).

FIG. 8. Overview of Reverse Phase High Pressure Liquid Chromatography(RP-HPLC). RP-HPLC is an analytical technique used for separating,characterizing, and quantifying analytes within a mixture. RP-HPLCrelies on an aqueous polar phase that is pressurized and pumped througha column filled with a non-polar stationary phase composed ofoctadecylcarbon chain (C18)-bonded silica. The mobile polar phasecarries the sample to the column where the analytes adsorb to thenon-polar stationary phase through hydrophobic interactions. Dependingon the relative affinity of the analyte between the stationary andmobile phases dictates the amount of time required for the analyte toelute from the column. Analytes with higher polarities will interactwith the column less, and therefore elute from the column faster. As theanalytes exit the column they can be detected by their UV absorbance, orif radiolabeled, by the in-line liquid scintillation analyzer.

FIG. 9. RP-HPLC calibration curve for progesterone. (Panel a) Molecularstructure of progesterone (PD). (Panel b)(Panel b) 3-D spectral datashowing progesterone's absorbance wavelength (λ_(max): 245 nm) andretention time (t_(r)=13.8 min). (Panel c) Serial 2-D chromatograms withincreasing concentrations of progesterone. (Panels d & e) Demonstrationof the sensitivity and quantitative ability in assessing the number ofmicrograms in a serial dilution of progesterone by RP-HPLC. Injectionvolumes of 80 μL from serial dilutions between 0.025 μg to 2.5 μgprogesterone resulted in a calibration curve with an R² of 1.

FIG. 10. RP-HPLC analysis of pUC19 transformed E. coli clarified lysateincubated with cholesterol (CL). (Panel a) Molecular structure of CL.Representative (Panel b)(Panel b) 2-D chromatogram (λ239 nm), (Panel c)C4-¹⁴C scintillation events, and (Panel d)(Panel d) 3-D chromatogramfrom pUC19 transformed bacterial lysate following incubation with 3.87μg (100 μM) cholesterol spiked with 20 nCi C4-14C labeled CL (λ_(max):<200 nm; t_(r)=38.9 min) for 24 hours. (Panel b)(Panel b) Analysis ofthe clarified lysate reveals E. coli lack the ability to metabolize CLinto new UV absorbing products within 24 hours. (Panel c) Analysis ofC4-¹⁴C scintillation events confirms this inability as the reduction ofC4-¹⁴C CL into downstream radiolabeled intermediates is not observed.Lastly, (Panel d)(Panel d) the 3-D chromatogram reinforces that while CLlacks UV absorbance within 200-300 nm, the clarified bacterial lysate isunable to metabolize CL into products that produce a UV absorbancebetween the 200-300 nm range following 24 hours of incubation. Together,the chromatograms demonstrate why E. coli are ideal for characterizingthe humanized cholesterol catabolizing enzymes in the presence of CL asa substrate.

FIG. 11. RP-HPLC analysis of pUC19 transformed E. coli clarified lysateincubated with cholestenone (CN). (Panel a) Molecular structure of CN.Representative (Panel b)(Panel b) 2-D chromatogram (λ239 nm), (Panel c)CN UV absorbance spectrum, and (Panel d)(Panel d) 3-D chromatogram frompUC19 transformed bacterial lysate following incubation with 3.85 μg(100 μM) cholestenone (λ_(max): 239 nm; t_(r)=36.9 min) for 24 hours.(Panel b)(Panel b) Analysis of the clarified lysate reveals E. coli lackthe ability to metabolize CN into new UV absorbing products within 24hours. (Panel c) Analysis of the CN UV absorbance shows the 36.9 minpeak has a λ_(max) of 239 nm, matching the CN analytical standard.Lastly, (Panel d)(Panel d) the 3-D chromatogram reinforces the emptyvector transformed bacterial lysate lacks the ability to metabolize CNinto products that produce a UV absorbance between the 200-300 nm rangefollowing 24 hours of incubation. Together, the chromatogramsdemonstrate why E. coli are ideal for characterizing the humanizedcholesterol catabolizing enzymes in the presence of CN as a substrate.

FIG. 12. RP-HPLC analysis of pUC19 transformed E. coli clarified lysateincubated with pregnenolone (PL). (Panel a) Molecular structure of PL.Representative (Panel b)(Panel b) 2-D chromatogram (λ245 nm), (Panel c)PL UV absorbance spectrum, and (Panel d) 3-D chromatogram from pUC19transformed bacterial lysate following incubation with 3.16 μg (100 μM)pregnenolone (λ_(max): <200 nm; t_(r)=15.5 min) for 24 hours. (Panelb)(Panel b) Analysis of the clarified lysate reveals E. coli lack theability to metabolize PL into new UV absorbing products within 24 hours.(Panel c) Analysis of the PL UV absorbance reveals the substratesmaximal UV absorbance is found below the 200 nm wavelength range.Lastly, (Panel d) the 3-D chromatogram reinforces the empty vectortransformed bacterial lysate lacks the ability to metabolize PL intoproducts that produce a UV absorbance between the 200-300 nm rangefollowing 24 hours of incubation. Together, the chromatogramsdemonstrate why E. coli are ideal for characterizing the humanizedcholesterol catabolizing enzymes in the presence of PL as a substrate.

FIG. 13. RP-HPLC analysis of pUC19 transformed E. coli clarified lysateincubated with progesterone (PD). (Panel a) Molecular structure of PD.Representative (Panel b)(Panel b) 2-D chromatogram (λ245 nm), (Panel c)C4-¹⁴C scintillation events, and (Panel d) 3-D chromatogram from pUC19transformed bacterial lysate following incubation with 3.14 μg (100 μM)progesterone spiked with 20 nCi C4-¹⁴C labeled PD (λ_(max): 245 nm;t_(r)=13.8 min) for 24 hours. (Panel b)(Panel b) Analysis of theclarified lysate reveals E. coli lack the ability to metabolize PD intonew UV absorbing products within 24 hours. (Panel c) Analysis of C4-¹⁴Cscintillation events confirms this inability as the reduction of theC4-¹⁴C PD substrates into downstream radiolabeled intermediates is notobserved. Lastly, (Panel d) the 3-D chromatogram reinforces the emptyvector transformed bacterial lysate lacks the ability to metabolize PDinto products that produce a UV absorbance between the 200-300 nm rangefollowing 24 hours of incubation. Together, the chromatogramsdemonstrate why E. coli are ideal for characterizing the humanizedcholesterol catabolizing enzymes in the presence of PD as a substrate.

FIG. 14. RP-HPLC analysis of cholestenone (CN) formation fromcholesterol (CL) utilization by E. coli clarified lysate expressinghumanized cholesterol dehydrogenase (CholD). (Panel a) Reaction overviewof CN formation from CLring-A3β-hydroxyloxidation by CholD.Representative (Panel b)(Panel b) 2-Dchromatogram (λ239 nm), (Panel c)C4-¹⁴C scintillation events, and (Panel d) 3-Dchromatogram from CholDbacterial lysate following incubation with 3.87 μg (100 μM) cholesterolspiked with 60 nCiC4-¹⁴C labeled CL (λ_(max):<200 nm; t_(r)=38.9 min)for 24 hours. (Panel b)(Panel b) Analysis of the CholDlysates howsreduction in CL and formation of CN(λ_(max):239 nm; t_(r)=36.9 min)within 24 hours. (Panel c) Analysis of C4-¹⁴C scintillation eventsconfirms that production of radiolabeled CN isconcomitant to thereduction of C4-¹⁴CCL. Lastly, (Panel d) the 3-Dchromatogram reinforcesthat while CL lacks UV absorbance within 200-300 nm, the 3β-oxidation ofCL produces a new peak with the same characteristic λ_(max) and t_(r) ofCN that is not observed in the control pUC19 lysate.

FIG. 15. RP-HPLC analysis of progesterone (PD) formation frompregnenolone (PL) utilization by E. coli clarified lysate expressinghumanized cholesterol dehydrogenase (CholD). (Panel a) Reaction overviewof PD formation from PL ring-A 3β-hydroxyl oxidation by CholD.Representative (Panel b)(Panel b) 2-D chromatogram (λ245 nm), (Panel c)PD UV absorbance spectrum, and (Panel d) 3-D chromatogram from CholDbacterial lysate following incubation with 3.16 μg (100 μM) pregnenolone(λ_(max): <200 nm; t_(r)=15.5 min) for 24 hours. (Panel b)(Panel b)Analysis of the CholD lysate shows formation of PD (λ_(max): 245 nm;t_(r)=13.8 min) within 24 hours. (Panel c) Analysis of the PD UVabsorbance spectrum shows the λmax of the 13.8 min peak is 245 nm.Lastly, (Panel d) the 3-D chromatogram reinforces that while PL lacks UVabsorbance within 200-300 nm, the 3β-oxidation of PL produces a new peakwith the same characteristic λ_(max) and t_(r) of PD that is notobserved in the control pUC19 lysate.

FIG. 16. RP-HPLC analysis of choleste-1,4-diene-3-one (CDN) formationfrom cholestenone (CN) utilization by E. coli clarified lysateexpressing humanized anoxic cholesterol metabolism B enzyme (acmB).(Panel a) Reaction overview of CDN formation from CN ring-A C1-C2desaturation by acmB. Representative (Panel b)(Panel b) 2-D chromatogram(λ241 nm), (Panel c) CDN UV absorbance spectrum, and (Panel d) 3-Dchromatogram from acmB bacterial lysate following incubation with 3.85μg (100 μM) cholestenone (λ_(max): 239 nm; t_(r)=36.9 min) for 24 hours.(Panel b)(Panel b) Analysis of the acmB lysate shows formation of CDN(λ_(max): 241 nm; t_(r)=29.8 min) within 24 hours. (Panel c) Analysis ofthe CDN UV absorbance spectrum shows the λ_(max)Of the 36.9 min peak is241 nm. Lastly, (Panel d) the 3-D chromatogram reinforces that acmB hasthe ability to desaturate the C1-C2 bond of CN by the formation of a newpeak with a unique λ_(max) and t_(r) that is not observed in the controlpUC19 lysate.

FIG. 17. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) utilization by E. coli clarified lysateexpressing humanized anoxic cholesterol metabolism B enzyme (acmB).(Panel a) Reaction overview of PDD formation from PD ring-A C1-C2desaturation by acmB. Representative (Panel b) 2-D chromatogram (λ245nm), (Panel c) C4-¹⁴C scintillation events, and (Panel d) 3-Dchromatogram from acmB bacterial lysate following incubation with 3.14μg (100 μM) progesterone spiked with 20 nCi C4-¹⁴C labeled PD (λ_(max):245 nm; t_(r)=13.8 min) for 24 hours. (Panel b) Analysis of acmB lysateshows reduction in PD and formation of PDD (λmax: 247 nm; t_(r)=10.0min) within 24 hours. (Panel c) Analysis of C4-¹⁴C scintillation eventsconfirms that production of radiolabeled PDD is concomitant to thereduction of C4-¹⁴C PD. Lastly, (Panel d) the 3-D chromatogramreinforces that acmB has the ability to desaturate the ring-A C1-C2 bondof PD to produce a new peak with a unique λ_(max) and t_(r) that is notobserved in the control pUC19 lysate.

FIG. 18. RP-HPLC analysis of the clarified lysate from E. coliexpressing humanized 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD)demonstrating a lack in the ability to form choleste-1,4-diene-3-one(CDN) from cholestenone (CN). (Panel a) Reaction overview of CDNformation from CN ring-A C1-C2 desaturation by Δ¹-KstD. Representative(Panel b) 2-D chromatogram (Δ 239 nm), (Panel c) CN UV absorbancespectrum, and (Panel d) 3-D chromatogram from Δ¹-KstD bacterial lysatefollowing incubation with 3.85 μg (100 μM) cholestenone (λ_(max): 239nm; t_(r)=36.9 min) for 24 hours. (Panel a) Analysis of the 2-Dchromatogram reveals Δ¹-KstD is unable to form CDN (λ_(max): 241 nm;t_(r)=29.8 min) from CN within 24 hours. (Panel b) Analysis of the CN UVabsorbance spectrum shows a detectable peak within 200-300 nm has at_(r) of 36.9 and a λ_(max) of 239 nm, matching the CN analyticalstandard. Lastly, (Panel c) the 3-D chromatogram reinforces that Δ¹-KstDlacks the ability to desaturate the C1-C2 bond of CN as the chromatogrammatches the control pUC19 lysate.

FIG. 19. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) utilization by E. coli clarified lysateexpressing humanized 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD). (Panel a)Reaction overview of PDD formation from PD ring-A C1-C2 desaturation byΔ¹-KstD. Representative (Panel b) 2-D chromatogram (λ245 nm), (Panel c)C4-14C scintillation events, and (Panel d) 3-D chromatogram from Δ¹-KstDbacterial lysate following incubation with 3.14 μg (100 μM) progesteronespiked with 20 nCi C4-14C labeled PD (λ_(max): 245 nm; t_(r)=13.8 min)for 24 hours. (Panel b) Analysis of Δ¹-KstD lysate shows reduction in PDand formation of PDD (λ_(max): 247 nm; t_(r)=10.0 min) within 24 hours.(Panel c) Analysis of C4-¹⁴C scintillation events confirms thatproduction of radiolabeled PDD is concomitant to the reduction of C4-¹⁴CPD. Lastly, (Panel d) the 3-D chromatogram reinforces that Δ¹-KstD hasthe ability to desaturate the ring-A C1-C2 of PD to produce a new peakwith a unique λ_(max) and t_(r) that is not observed in the controlpUC19 lysate.

FIG. 20. RP-HPLC analysis of 9-hydroxycholeste-4-ene-3-one (9-OHCN)formation from cholestenone (CN) utilization by E. coli clarified lysateexpressing humanized 3-ketosteroid 9α-hydroxylase (KshAB). (Panel a)Reaction overview of 9-OHCN formation from CN ring-B C9 hydroxylation byKshAB. Representative (Panel b) 2-D chromatogram (λ239 nm), (Panel c)9-OHCN UV absorbance spectrum, and (Panel d) 3-D chromatogram from KshABbacterial lysate following incubation with 3.85 μg (100 μM) cholestenone(λ_(max): 239 nm; t_(r)=36.9 min) for 24 hours. (Panel b) Analysis ofKshAB lysate shows a slight reduction in CN and small formation of9-OHCN (λ_(max): 239 nm; t_(r)=8.9 min) within 24 hours. (Panel c)Analysis of the 9-OHCN UV absorbance spectrum shows the λ_(max)Of the8.9 min peak is 239 nm. Lastly, (Panel d) the 3-D chromatogramreinforces that KshAB has the ability to hydroxylate C9 of the CN ring-Bto produce a new peak with a unique t_(r) that is not observed in thecontrol pUC19 lysate.

FIG. 21. RP-HPLC analysis of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)formation from progesterone (PD) utilization by E. coli clarified lysateexpressing humanized 3-ketosteroid 9α-hydroxylase (KshAB). (Panel a)Reaction overview of 9-OHPD formation from PD ring-B C9 hydroxylation byKshAB. Representative (Panel b) 2-D chromatogram (λ245 nm), (Panel c)C4-¹⁴C scintillation events, and (Panel d) 3-D chromatogram from KshABbacterial lysate following incubation with 3.14 μg (100 μM) progesteronespiked with 20 nCi C4-¹⁴C labeled PD (λ_(max): 245 nm; t_(r)=13.8 min)for 24 hours. (Panel b) Analysis of KshAB lysate shows reduction in PDand formation of 9-OHPD (λ_(max): 245 nm; t_(r)=5.2 min) within 24hours. (Panel c) Analysis of C4-¹⁴C scintillation events confirms thatproduction of radiolabeled 9-OHPD is concomitant to the reduction ofC4-¹⁴C PD. Lastly, (Panel d) the 3-D chromatogram reinforces that KshABhas the ability to hydroxylate C9 of the PD ring-B to produce a new peakwith a unique t_(r) that is not observed in the control pUC19 lysate.

FIG. 22. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationfrom progesterone (PD) utilization by E. coli clarified lysatesexpressing humanized anoxic cholesterol metabolism B enzyme (acmB) and3-ketosteroid 9α-hydroxylase (KshAB). (Panel a) Reaction overview of3-HSP formation from PD ring-A C1-C2 desaturation and ring-B C9hydroxylation by acmB and KshAB, respectively. Representative (Panel b)2-D chromatogram (λ280 nm), (Panel c) C4-¹⁴C scintillation events, and(Panel d) 3-D chromatogram from acmB and KshAB bacterial lysatesfollowing incubation with 6.28 μg (100 μM) progesterone spiked with 40nCi C4-¹⁴C labeled PD (λ_(max): 245 nm; t_(r)=13.8 min) for 24 hours.(Panel b) Analysis of acmB and KshAB lysates shows complete reduction inPD and formation of 3-HSP (λ_(max): 280 nm; t_(r)=7.2 min) within 24hours. (Panel c) Analysis of C4-¹⁴C scintillation events confirms thatproduction of radiolabeled 3-HSP is concomitant to the reduction ofC4-¹⁴C PD. Lastly, (Panel d) the 3-D chromatogram reinforces thattogether, acmB and KshAB have the ability to desaturate the ring-A C1-C2bond and hydroxylate the ring-B C9 of PD to produce a new peak with aunique λ_(max) and t_(r) that is not observed in the control pUC19lysate.

FIG. 23. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationfrom progesterone (PD) utilization by E. coli clarified lysatesexpressing humanized 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) and3-ketosteroid 9α-hydroxylase (KshAB). (Panel a) Reaction overview 3-HSPformation from PD ring-A C1-C2 desaturation and ring-B C9 hydroxylationby Δ¹-KstD and KshAB, respectively. Representative (Panel b) 2-Dchromatogram (λ280 nm), (Panel c) C4-¹⁴C scintillation events, and(Panel d) 3-D chromatogram from Δ¹-KstD and KshAB bacterial lysatesfollowing incubation with 6.28 μg (100 μM) progesterone spiked with 40nCi C4-¹⁴C labeled PD (λ_(max): 245 nm; t_(r)=13.8 min) for 24 hours.(Panel b) Analysis of Δ¹-KstD and KshAB lysates shows complete reductionin PD and formation of 3-HSP (λ_(max): 280 nm; t_(r)=7.2 min) within 24hours. (Panel c) Analysis of C4-¹⁴C scintillation events confirms thatproduction of radiolabeled 3-HSP is concomitant to the reduction ofC4-¹⁴C PD. Lastly, (Panel d) the 3-D chromatogram reinforces thattogether, Δ¹-KstD and KshAB have the ability to desaturate the ring-AC1-C2 bond as well as hydroxylate the ring-B C9 of PD to produce a newpeak with a unique λ_(max) and t_(r) that is not observed in the controlpUC19 lysate.

FIG. 24. RP-HPLC analysis of3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one (3-HSC) formationfrom cholesterol (CL) utilization by E. coli clarified lysatesindependently expressing humanized cholesterol dehydrogenase (CholD),anoxic cholesterol metabolism B enzyme (acmB) and 3-ketosteroid9α-hydroxylase (KshAB). (Panel a) Reaction overview 3-HSC formation fromCL ring-A 3β-hydroxyl oxidation, ring-A C1-C2 desaturation, and ring-BC9 hydroxylation by CholD, acmB, and KshAB, respectively. Representative(Panel b) 2-D chromatogram (λ239 nm), (Panel c) 2-D chromatogram (λ280nm), (Panel d) C4-¹⁴C scintillation events, and (Panel d) 3-Dchromatogram from a mixed reaction of CholD, acmB, and KshAB bacteriallysates incubated with 11.60 μg (100 μM) cholesterol spiked with 100 nCiC4-¹⁴C labeled CL (λ_(max): <200 nm; t_(r)=38.9 min) for 24 hours.(Panels b & c) Analysis of CholD, acmB, and KshAB lysates showsformation of cholestenone (CN) (λ_(max): 239 nm; t_(r)=36.0 min),choleste-1,4-diene-3-one (CDN) (λ_(max): 241 nm; t_(r)=29.5 min), and3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one (3-HSC) (λ_(max):280 nm; t_(r)=5.3 min) within 24 hours. (Panel d) Analysis of C4-¹⁴Cscintillation events confirms production of radiolabeled 3-HSC isconcomitant to the reduction of C4-¹⁴C CL. Lastly, (Panel e) the 3-Dchromatogram reinforces that when combined, CholD, acmB, and KshAB equipthe bacterial lysates with the ability to oxidize the 3β-hydroxyl to a3-ketone, desaturate the C1-C2 bond of ring-A, and hydroxylate thering-B C9 of CL, respectively. The presence of all three humanizedenzymes equip the bacterial lysates with the ability to produce 3-HSC, anovel compound having a unique λ_(max) and t_(r) that is not observed inthe control pUC19 lysate incubated with CL.

FIG. 25. RP-HPLC analysis of E. coli clarified lysates independentlyexpressing humanized cholesterol dehydrogenase (CholD), 3-ketosteroidΔ¹-dehydrogenase (Δ¹-KstD) and 3-ketosteroid 9α-hydroxylase (KshAB) areunable to produce 3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one(3-HSC) from cholesterol (CL). (Panel a) Reaction overview 3-HSCformation from CL ring-A 3β-hydroxyl oxidation, ring-A C1-C2desaturation, and ring-B C9 hydroxylation by CholD, Δ¹-KstD, and KshAB,respectively. Representative (Panel b) 2-D chromatogram (λ239 nm),(Panel c) C4-¹⁴C scintillation events, and (Panel d) 3-D chromatogramfrom a mixed reaction of CholD, Δ¹-KstD, and KshAB bacterial lysatesincubated with 11.60 μg (100 μM) cholesterol spiked with 100 nCi C4-¹⁴Clabeled CL (λ_(max): <200 nm; t_(r)=38.9 min) for 24 hours. (Panel b)Analysis of CholD, Δ¹-KstD, and KshAB lysates shows formation ofcholestenone (CN) (λ_(max): 239 nm; t_(r)=36.0 min) and9-hydroxycholeste-4-ene-3-one (9-OHCN) (λ_(max): 239 nm; t_(r)=8.9 min),but not choleste-1,4-diene-3-one (CDN) (λ_(max): 241 nm; t_(r)=29.5 min)or 3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one (3-HSC)(λ_(max): 280 nm; t_(r)=5.3 min) within 24 hours. (Panel c) Analysis ofC4-¹⁴C scintillation events confirms Δ¹-KstD lacks the ability todesaturate CN due to the presences of the side chain. Lastly, (Panel d)the 3-D chromatogram reinforces that when combined, CholD and KshABequip the bacterial lysates with the ability to oxidize the 3β-hydroxylto a 3-ketone and hydroxylate the ring-B C9 of CL, respectively.However, due to the presence of the C17 sidechain, Δ¹-KstD is unable todesaturate the C1-C2 bond of ring-A and thus, the combined bacteriallysates lack the ability to form 3-HSC.

FIG. 26. Cholestenone dose response curve with Hep3B cells. Hep3B cellswere seeded (6×10⁴ cells/well) to reach 90% confluency on day oftreatment. Cells were incubated in 200 μL media A containing 30-110 μMcholestenone for 72 hours. After 48 hours of cholestenone treatment, 120μM of resazurin was added to each well for an additional 24 hours.Following incubation, the fluorescent intensity of each well wasmeasured at 540±25 ex and 620±40 emwith a BioTeKSynergy 2 plate reader.The mean fluorescent intensity from each concentration of cholestenonewas graphed as a percent of the maximum fluorescent intensity. Datarepresents an N of 1 with 8 replicates. Error bars indicate the standarderror of the mean.

FIG. 27. BODIPY 493/503 stained U-937 derived macrophages and foamcells. U-937 Monocytes were differentiated with 200 nM PMA for 48 hours.PMA was removed and cells were allowed to continue differentiating forthree additional days. Five day old macrophages were incubated with 50μg acLDL for 24 hours. Cells were stained with 1 μg/mL BODIPY 493/503for 30 min at 37° C. for 30 min and imaged with a Nikon A1confocalmicroscope.

FIG. 28. RP-HPLC analysis of progesterone (PD) product formation fromC4-¹⁴C cholesterol labeled LDLs by P450-FdxR-Fdx-P2A-HSD2 expressingU-937-derived macrophages. Representative 2-D chromatograms of (Panels a& b) λ245 nm, (Panels c & d) C4-¹⁴C scintillation events, and (Panels e& f) 3-D spectral data from (Panels a, c, & e) control macrophages and(Panels b, d, & f) P450-FdxR-Fdx-2A-HSD2 expressing macrophagesincubated with 50 μg C4-¹⁴C cholesterol labeled LDLs (163 nCi C4-14Ccholesterol) for 72 hours. Analysis at (Panel b) λ245 nm revealsP450-FdxR-Fdx-2A-HSD2 macrophages are equipped with the ability tohydrolyze the cholesterol side chain and oxidize the 3β-hydroxyl to a3-ketone forming PD (t_(r)=13.8 min, λ_(max)245 nm) following 72 hoursincubation. In contrast, (Panel a) control macrophages lack the abilityto convert cholesterol to progesterone at an appreciable amount.

FIG. 29. RP-HPLC analysis of progesterone (PD) product formation frompregnenolone (PL) by P450-FdxR-Fdx 2A HSD2 expressing U-937-derivedmacrophages. Representative 2-D chromatograms of (Panels a & b) λ245 nm,and (Panels c & d) 3-D spectral data from (Panels a & c) controlmacrophages and (Panels b & d) P450-FdxR-Fdx-2A-HSD2 expressingmacrophages incubated with 15.8 μg pregnenolone (PL) (λ_(max): <200 nm;t_(r)=15.5 min) for 72 hours. Analysis at (Panels b & d) λ245 nm revealsP450-FdxR-Fdx-2A-HSD2 macrophages are equipped with the ability tooxidize the 3β-hydroxyl to a 3-ketone forming PD (t_(r)=13.8 min,λ_(max24)5 nm) following 72 hours incubation. In contrast, (Panels a &c) control macrophages lack the ability to convert PL to PD.

FIG. 30. Purification outline for Δ¹-KstD. The details are described inthe section of Materials and Methods, below at paragraphs[00304]-[00351].

FIG. 31. Chromatogram from Δ¹-KstD isolation using immobilized metalaffinity chromatography (IMAC). Representative 2-D chromatogram showingthe elution profile of the clarified lysate from E. coli expressing theHP-Thioredoxin (HP-THX) Δ¹-KstDfusion protein. The lysate was loadedonto a 5 mL G.E. HiTrap nickel chelating column using a 50 mL superloop. Protein elution was monitored at λ280 nm and is represented by theblue line. A linear gradient of imidazole, represented by the gold line,was used to elute HP-THX Δ¹-KstD. The run begins with a 20 mM imidazoleisocratic wash for 150 mL. The first 50 mLs of eluate contained the flowthrough, and was followed by 100 mL wash. The gradient begins with a 50mM imidazole step into a 50 mL linear gradient to 200 mM imidazole. Thecolumn was washed with 200 mM imidazole for an additional 50 mL beforereturning to 20 mM imidazole for 20 mL.

FIG. 32. Nitrotetrazolium blue (NTB) reaction mechanism. The NTB assayis an indirectly coupled redox reaction that allows the assessment ofthe relative dehydrogenase activity found in each IMAC fraction. Thereaction proceeds with the removal of two high energy electrons fromprogesterone (PD) by Δ¹-KstD to form pregn-1,4-diene-3,20-dione (PDD).The electrons are passed from the FADH of Δ¹-KstD to the intermediateelectron acceptor, phenazine methylsulfate (PMS). PMS relays theelectrons to NTB where the center tetrazolium ring is reduced toformazan. The reaction causes NTB, which in the oxidized state forms asoluble yellow solution, to transition into an insoluble purpleprecipitate at sites where dehydrogenation is occurring. The assay wasadapted to be used in a Native-PAGE format to assess the relativedehydrogenase activity of each IMAC fraction.

FIG. 33. In-gel nitrotetrazolium blue (NTB) activity assay of fractionscollected from the Δ¹-KstD isolation by immobilized metal affinitychromatography. Protein samples of the fractions collected from theHiTrap nickel chelating column were mixed with an equal volume of 2×native sample buffer. Equivalent volumes of each fraction were separatedusing Native-PAGE on a 10% polyacrylamide gel. Δ¹-KstD activity wasvisualized by NTB staining for 5 min. NTB staining buffer consists of160 nM PMS, 80 nM NTB, 1.5 nM progesterone (PD) in 66.7 mM Tris.Equivalent volumes (5 μL) from the lysate (L), fractions 2, 4, 6, 13,19, 20, 21, 22, 23, 24, 25, 26, and 27 are shown.

FIG. 34. Coomassie blue stained SDS-PAGE of fractions collected fromΔ¹-KstD isolation by immobilized metal affinity chromatography. Proteinsamples of the fractions collected from the HiTrap nickel chelatingcolumn were mixed with an equal volume of 2× Laemmli sample buffer,boiled for 5 min, and spun at 15,000×g for 10 min at 4° C. Equivalentvolumes of each fraction were separated using SDS-PAGE on a 10%polyacrylamide gel and visualized by Coomassie blue staining. Separatedproteins from the lysate (L), fractions 2, 4, 6, 13, 19, 20, 21, 22, 23,24, 25, 26, and 27 are shown. Elution fraction 21 was selected forfurther characterization.

FIG. 35. Western blot analysis of Δ¹-KstD isolation from fractionscollected by immobilized metal affinity chromatography. Protein samplesof the fractions collected from the HiTrap nickel chelating column weremixed with an equal volume of 2×SDS sample buffer, boiled for 5 min, andspun at 15,000×g for 10 min at 4° C. Equivalent volumes of each fractionwere separated using SDS-PAGE on a 10% polyacrylamide gel, transferredto a PVDF membrane, and probed with anti-FLAG (1:1000). ECL anti-mouseIgGsecondary antibody conjugated to HRP (1:10,000) and SuperSignalWestFemtoSubstrate was used for detection. Separated proteins from thelysate (L), fractions 2, 4, 6, 13, 19, 20, 21, 22, 23, 24, 25, 26, and27 are shown. Elution fraction 21 was selected for furthercharacterization.

FIG. 36. Yield and purity of isolated Δ¹-KstD. Concentration ofpartially purified Δ¹-KstDwas determined to be 0.385 mg/mL with 79.6%purity determined by densitometry (ImageJ) of elution fraction (EF) 21from the coomassie stained SDS-PAGE.

FIG. 37. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) desaturation by isolated Δ¹-KstD. Representative2-D chromatograms and 3-D spectral data from (Panels a & b) 6.29 μg PDanalytical standard and (Panels c & d) an isolated Δ¹-KstD reactioncontaining 770 ng of enzyme incubated with 6.29 μg PD for four hours at37° C. (Panels c & d) Results reveal a diminished absorbance maximum atthe retention time typical of the substrate PD (λ_(max): 245 nm,t_(r)=13.8 min) and formation of the product PDD (λ_(max): 247 nm,t_(r)=10.0 min) by RP-HPLC-based analysis. Within four hours, Δ¹-KstDconverted 90% of the substrate PD to PDD. This data demonstrates thatthe enzyme isolated from IMAC is in fact Δ¹-KstD and is highly active.

FIG. 38. Resazurin reaction mechanism. Resazurin, a weakly fluorescentredox dye, is irreversibly reduced upon accepting electrons removed fromthe desaturation of progesterone's (PD) ring-A C1-C2 bond by Δ¹-KstD.Resazurin's center ring has an electronegative oxygen pulling the lonepair electrons from nitrogen, forming a zwitterion. Once the oxygen isreduced by the electrons released from the desaturation of progesterone(PD), water acts as a leaving group freeing the lone pair electrons ofnitrogen to form the highly fluorescent compound, resorufin. Unlikeindirectly coupled assays that use NAD+, diaphorase, or otherintermediate electron acceptors to relay electrons to the redox dye,resazurin is a directly coupled reaction. This assay is unique in thatresazurin is able to directly accept the liberated protons, allowing oneto measure the rate of substrate desaturation by Δ¹-KstD through theformation of the fluorescent product, resorufin.

FIG. 39. Resorufin/resazurin standard curve. To best represent thereaction that occurs in the resazurin assay during the desaturation ofprogesterone (PD) by Δ¹-KstD, several concentrations of resorufin wereadded to inversely proportional concentrations of resazurin. Thesevalues were expressed as a percent of resorufin, totaling to 20 μMresorufin and resazurin. The data shows a linear increase influorescence with increasing concentration of resorufin in the presenceof decreasing concentrations of resazurin.

FIG. 40. Δ¹-KstD enzyme titration curves. Effect of Δ¹-KstD enzymeconcentration (2.1 nM; square, 1.6 nM; circle, 1.1 nM; upward facingtriangle, 0.55 nM; downward facing triangle, 0.37 nM; diamond, 0.19 nM;left facing triangle, and 0.05 nM; right facing triangle) on fluorescentsignal (RFU) with respect to time using fixed concentrations ofprogesterone (20 μM) as substrate and resazurin (20 μM) as thefluorescent electron acceptor. Resorufin fluorescence was measured at 17sec intervals for 3.5 min. All seven concentrations of Δ¹-KstDdemonstrate a linear increase in fluorescence over 3.5 min in thepresence of 20 μM progesterone and 20 μM resazurin. Each enzymetitration curve was made with an N of 1 in triplicate.

FIG. 41. 1.6 nM Δ¹-KstD enzyme progress curves. Reaction progress curvesat fixed concentrations of Δ¹-KstD (1.6 nM), fixed concentrations ofresazurin (20 μM), and varying concentrations of progesterone (40 μM;square, 30 μM; circle, 20 μM; upward facing triangle, 10 μM; downwardfacing triangle, 5 μM; diamond, 2.5 μM; left facing triangle, and 1 μM;right facing triangle). Resorufin fluorescence was measured at 17 secintervals for 10 minutes. Of the three enzyme progress curves, 1.6 nMΔ¹-KstD demonstrated the lowest linear increase in fluorescence over 10min in the presence of varying concentrations of progesterone. Eachenzyme titration curve was made with an N of 1 with 8 replicates. Errorbars indicate the standard error of the mean.

FIG. 42. 1.1 nM Δ¹-KstD enzyme progress curves. Reaction progress curvesat fixed concentrations of Δ¹-KstD (1.1 nM), fixed concentrations ofresazurin (20 μM), and varying concentrations of progesterone (40 μM;square, 30 μM; circle, 20 μM; upward facing triangle, 10 μM; downwardfacing triangle, 5 μM; diamond, 2.5 μM; left facing triangle, and 1 μM;right facing triangle). Resorufin fluorescence was measured at 17 secintervals for 10 minutes. The 1.1 nM Δ¹-KstD enzyme progress curvesdemonstrated an improvement in fluorescent linearity, as compared to the1.6 nM Δ¹-KstD enzyme progress curves. Each enzyme titration curve wasmade with an N of 1 with 8 replicates. Error bars indicate the standarderror of the mean.

FIG. 43. 0.55 nM Δ¹-KstD enzyme progress curves. Reaction progresscurves at fixed concentrations of Δ¹-KstD (0.55 nM), fixedconcentrations of resazurin (20 μM), and varying concentrations ofprogesterone (40 μM; square, 30 μM; circle, 20 μM; upward facingtriangle, 10 μM; downward facing triangle, 5 μM; diamond, 2.5 μM; leftfacing triangle, and 1 μM; right facing triangle). Resorufinfluorescence was measured at 17 sec intervals for 10 minutes. Of thethree enzyme progress curves, 0.55 nM Δ¹-KstD demonstrated the highestlinearity in fluorescence over the 10 min measurement. Each enzymetitration curve was made with an N of 1 with 8 replicates. Error barsindicate the standard error of the mean.

FIG. 44. Kinetic analysis of progesterone C1-C2 A-ring desaturation byΔ¹-KstD. Resorufin fluorescence was measured for each reaction at 17second intervals for 10 min. Initial velocities of the reactions weredetermined from the linear portion of the 0.55 nM Δ¹-KstD progresscurves by least squares analysis and plotted against the substrateconcentration. Data are shown for all Δ¹-KstD progress curves (1.6 nM;circle, 1.1 nM; triangle, and 0.55 nM; square) which were made with 8replicates at each indicated concentration of progesterone and fixedconcentrations of resazurin (20 μM). Error bars indicate the standarderror of the mean. K_(m)(8.3+/−0.5 μM) and V_(max)(2.2+/−0.05 RFU/sec)were determined by fitting the data to the Michaelis-Menten equation.

FIG. 45. Δ¹-KstD substrate specificity screen. Substrate preference ofΔ¹-KstD was assessed with 21 cholesterol derivatives (pregnane-,adrostane-, and cholestane-based derivatives). Reaction mixturescontaining 5.35 nM Δ1-KstD and 0.1 mg/mL BSA (dispensed with a PDsyringe) were equilibrated for 30 sec before the reaction was initiatedby adding 20 μM resazurin and 20 μM of the steroid substrate. Of thetwenty-one substrates screened, eight were found to substrates forΔ¹-KstD. Data reveals Δ¹-KstD requires a 3-ketone on ring-A; Δ¹-KstDspecificity exceeds that of the previously established substrate,androstenedione; however, Δ¹-KstD lacks the capability to utilizesubstrates with long, alkyl C17 side chains. Assay was performed with anN of 1 in quadruplicate.

FIG. 46. Δ¹-KstD preferred substrates. (Panels a-i) Representativestructures of eight substrates Δ¹-KstD demonstrated high activity withfrom the substrate specificity screen. (Panel a) pregn-4-ene-3,20-dione(progesterone), (Panel b) 4-pregnen-17-ol-3,20-dione(17-hydroxyprogesterone), (Panel c) 4-pregnen-21-ol-3,20-dione(11-deoxycortico-sterone), (Panel d) 4-androsten-17β-ol-3-one(testosterone), (Panel e) 4-pregnen-17α,21-diol-3,11,20-trione(cortisone), (Panel f) 4-androsten-3,17-dione (androstenedione), (Panelg) 7α-acetylthio-3-oxo-17α-pregn-4-ene-21,17-carbolactone(spironolactone), (Panel h) 5α-androstan-17β-ol-3-one(dihydrotestosterone), (Panel i) 17β-hydroxy-4-androsten-3-one17-enanthate (testosterone enanthate).

FIG. 47. Poor substrates for Δ¹-KstD. (Panels a-i) Representativestructures of twelve substrates from the substrate specificity screenΔ¹-KstD demonstrated little to no activity against. (Panel a)5α-androstan-3α-ol-17-one (androsterone), (Panel b)(11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione (hydrocortisone), (Panelc) 11β,17α,21-trihydroxy-4-pregnene-3,20-dione 21-hemisuccinate sodiumsalt (hydrocortisone 21-hemisuccinate), (Panel d)11β-(4-dimethylamino)phenyl-173-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one(mifepristone), (Panel e) 3β-hydroxy-5-cholestene (cholesterol), (Panelf) 3β-hydroxypregn-5-en-20-one (pregnenolone), (Panel g)5-androsten-3β-ol-17-one (dehydroepiandrosterone), (Panel h) (11β)-11,21-dihydroxypregn-4-ene-3, 20-dione(corticosterone), (Panel i)4-Cholesten-7b-ol-3-one (7β-hydroxycholestenone), (Panel j)(11β)-11,17,21-trihydroxypregna-1,4-diene-3,20-dione (prednisolone),(Panel k) choleste-4-ene-3-one (cholestenone), (Panel l)11β,21-dihydroxy-3,20-dioxopregn-4-en-18-al (aldosterone).

FIG. 48. Δ¹-KstDKozak Repair using Gibson Assembly. Δ¹-KstD's Kozakconsensus sequence was repaired using Gibson Assembly and synthetic DNA.The starting vector was linearized by double restriction enzyme digestto remove the attB1 site, TEV site, 6× His tag, Kozak consensussequence, tetracysteine tag, and Flag tag. The nucleotide sequence wasreplaced using synthetic DNA encoding the attB1 site, a new Kozakconsensus sequence, and a Flag tag flanked by 40 bp of homology to thebackbone vector. The vector was reassembled using Gibson assembly asdescribed in methods.

FIG. 49. Western blot analysis of Hep3B cells expressing CMV or PGKdriven Δ¹-KstD. Cells were transduced with increasing titers oflentiviral particles encoding Δ¹-KstD. Cells expressing Δ¹-KstD wereselected for using blasticidin(CMV) or hygromycin (PGK) antibiotic.Cells were passed into 60 mm dishes, grown to confluency, and collectedby scraping in 500 μL RIPA buffer. Cells were mechanically lysedon iceusing a syringe with a 27 gauge needle. Protein samples were mixed withan equal volume of 2× Laemmli sample buffer, boiled for 5 min, and spunat 15,000×g for 10 min at 4° C. Protein samples (25 μg) were separatedusing SDS-PAGE on a 10% polyacrylamide gel, transferred to a PVDFmembrane, and probed with anti-FLAG (1:1000). ECL anti-mouseIgGsecondary antibody conjugated to HRP (1:10,000) and SuperSignalWestFemtoSubstrate was used for detection. Samples include threerepresentative titers from both CMV and PGK driven Δ¹-KstD Hep3B cells,negative control Hep3B lysate (−C), empty lane (Panel E), and theisolated HP-THX Δ¹-KstD as a positive control (+C).

FIG. 50. RP-HPLC analysis of progesterone (PD) spiked with 100 nCiC4-¹⁴C labeled PD analytical standard. Representative (Panel a) 2-Dchromatogram at λ245 nm, (Panel b) C4-¹⁴C scintillation events, and(Panel c) 3-D spectral data of an 80 μL injection of 15.7 μg PD spikedwith 100 nCi C4-¹⁴C labeled PD in 500 μL HPLC running buffer 2. RP-HPLCanalysis reveals the PD substrate has a λ_(max) of 245 nm and a 13.8 minretention time (t_(r)).

FIG. 51. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) C1-C2 ring-A desaturation by Hep3B Δ¹-KstD cells.Representative 2-D chromatograms (λ245 nm; time points: 24, 48, and 72hours) from (Panel a) Hep3B control and (Panel b) Hep3B Δ¹-KstD cellsincubated with 15.7 μg (10 μM) progesterone spiked with 100 nCi C4-¹⁴Clabeled PD (t_(r)=13.8 min). Analysis of Hep3B Δ¹-KstD cells showsformation of PDD (t_(r)=10.0 min, λ_(max) 247 nm) throughout the 72 hourtime course. In contrast, control Hep3B cells lack the ability todesaturate the C1-C2 bond of the pregnane A-ring, as no 10.0 min peak isobserved.

FIG. 52. Quantitative analysis of pregn-1,4-diene-3,20-dione (PDD)product formation from progesterone (PD) C1-C2 ring A desaturation byHep3B Δ¹-KstD cells. Bar graphs representing the measured area under thecurve (AUC) of (Panels a & c) Hep3B control and (Panels b & d) Hep3BΔ¹-KstD cells incubated with 15.7 μg (10 μM) progesterone spiked with100 nCi C4-¹⁴C labeled PD. The AUC of PD (Panels a & b) and PDD (Panelsc & d) were measured at λ245 nm from 2-D chromatograms at time points:24, 48, and 72 hours. Quantitative analysis of PDD AUC reveals Hep3BΔ¹-KstD cells are equipped with the ability to desaturate the ring-AC1-C2 bond of PD to form the product PDD. However, Hep3B control cellslack this metabolic capability.

FIG. 53. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) utilization by Hep3B Δ¹-KstD cells.Representative 2-D chromatograms of C4-¹⁴C scintillation events at 24,48, and 72 hour time points from (Panel a) Hep3B control and (Panel b)Hep3B Δ¹-KstD cells incubated with 15.7 μg (10 μM) progesterone spikedwith 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min). Analysis of Hep3Bcontrol and Δ¹-KstD cells reveal reduction in C4-¹⁴C PD scintillationevents over the 72 hour time course for both group of cells. Reductionin the amount of PD in both groups is partially due to unidentifiedendogenous activity (EA) resulting in the accumulation of a new productpeak identified by C4-¹⁴C scintillation events (t_(r)=5.2 min). However,this new peak lacks an observable absorbance between 200-300 nm. Moreimportantly, concomitant to PD catabolism in Hep3B Δ¹-KstD cells, PDD(t_(r)=10.0 min) accumulates over the 72 hour time course as observed bya new peak forming with a λ_(max)Of 247 nm and containing C4-¹⁴Cscintillation events. Hep3B control cells lack the ability to catabolizePD to PDD, as seen by the absence of a peak with a 10.0 min retentiontime.

FIG. 54. Quantitative analysis of C4-¹⁴C scintillation events ofpregn-1,4-diene-3,20-dione (PDD) product formation from progesterone(PD) C1-C2 ring A desaturation by Hep3B Δ¹-KstD cells. Bar graphsrepresenting the measured counts under the curve (CUC) of Hep3B control(Panels a & c) and Hep3B Δ¹-KstD (Panels b & d) cells incubated with15.7 μg (10 μM) progesterone spiked with 100 nCi C4-¹⁴C labeled PD. TheCUC of PD (Panels a & b) and PDD (Panels c & d) were measured at 24, 48,and 72 hour time points. Quantitative analysis of PDD CUC reveals Hep3BΔ¹-KstD cells are equipped with the ability to desaturate the PD A-ringC1-C2 bond to form the product PDD, whereas, Hep3B control cells lackthis metabolic capability.

FIG. 55. RP-HPLC analysis of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)substrate. (Panel a) Representative 2-D chromatogram at λ245 nm from an80 μL injection of 17 μg 9-OHPD in 500 μL HPLC running buffer 2. 9-OHPDwas produced and isolated from bacterial KshAB lysate and used assubstrate for Hep3B Δ¹-KstD cells to determine whether cells equippedwith the ability to desaturate C1 and C2 of the pregnaneA-ring canproduce the ring opened compound,3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP). The 9-OHPDsubstrate standard in has a λ_(max) of 245 nm and a 5.2 min retentiontime (t_(r)). (Panel b) Representative HPLC chromatogram showing 9-OHPD(λ_(max): 245 nm; t_(r)=5.2 min) utilization in control cell lysates athour 0. (Panel c) 3-D chromatogram showing the spectral data (λ₃₀₀₋₂₀₀nm) plotted against time and absorption (mAU) of the sample run shown in(Panel b). (Panel d) Representative HPLC chromatogram showing the lackof 3-HSP (λ_(max): 280 nm; t_(r)=7.2 min) at 0 hours in control celllysates.

FIG. 56. Cholestane ring opening in human cells. In Hep3B Δ¹-KstD cells,catabolism of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD) forms3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP).Representative 2-D chromatograms (λ245 nm; time points: 2, 12, 36, and60 hours) from (Panel a) Hep3B control and (Panel b) Hep3B Δ¹-KstD cellsincubated with 17 μg (10 μM) 9-hydroxypregn-4-ene-3,20-dione (9-OHPD;t_(r)=5.2 min) produced and isolated from bacterial KshAB lysate.Analysis of Hep3B Δ¹-KstD cells shows reduction in 9-OHPD over the 72hour time course. In contrast, Hep3B control cells lack the metaboliccapability to catabolize 9-OHPD resulting in the substrates retentionover the time course.

FIG. 57. Cholestane ring opening in human cells. In Hep3B Δ¹-KstD cells,catabolism of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD) forms3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP). RP-HPLCanalysis of 3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP)formation by Hep3B Δ¹-KstD cells. Representative 2-D chromatograms (λ280nm; time points: 2, 12, 36, and 60 hours) from (Panel a) Hep3B controland (Panel b) Hep3B Δ¹-KstD cells incubated with 17 μg (10 μM)9-hydroxypregn-4-ene-3,20-dione (9-OHPD; t_(r)=5.2 min) produced andisolated from bacterial KshAB lysate. Analysis of Hep3B Δ¹-KstD cellsshows the formation of 3-HSP (t_(r)=7.2 min) concomitant to thereduction of 9-OHPD throughout the 72 hour time course. In contrast,control Hep3B cells lack the metabolic capability to catabolize 9-OHPD,and thus are unable to produce 3-HSP.

FIG. 58. Quantitative analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from 9-hydroxypregn-4-ene-3,20-dione (9-OHPD) C1-C2desaturation by Hep3B Δ¹-KstD cells. Bar graphs representing themeasured area under the curve (AUC) of (Panels a & b) 9-OHPD and (Panelsc &d) 3-HSP from (Panels a & c) Hep3B control and (Panels b & d) Hep3BΔ¹-KstD cells following incubation with 17 μg (10 μM) 9-OHPD producedand isolated from bacterial KshAB lysate. The AUC of 9-OHPD and 3-HSPwere measured at λ245 nm and λ280 nm, respectively, from 2-Dchromatograms at time points: 0, 2, 4, 8, 12, 24, 36, 48, 60, and 72hours. Quantitative analysis of 9-OHPD AUC reveals (Panel b) Hep3BΔ¹-KstD cells are equipped with the ability to catabolize the substrateas seen by its reduction and exhaustion over the 72 hour time course.Conversely, (Panel a) Hep3B control cells lack the metabolic ability tomodify the substrate, as seen by the retention of 9-OHPD throughout thetime course. Quantitative analysis of 3-HSP AUC reveals (Panel d) Hep3BΔ¹-KstD cells are equipped with the ability to desaturate C1-C2 bond of9-OHPD to form the ring opened product, 3-HSP. Conversely, (Panel c)Hep3B control cells lack the metabolic ability to catabolize 9-OHPD andtherefore are unable to produce 3-HSP.

FIG. 59. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) formationfrom progesterone (PD) by U-937 Δ¹-KstD cells. Representative 2-Dchromatograms of (Panels a & b) λ245 nm, (Panels c & d) C4-¹⁴Cscintillation events, and (Panels e & f) 3-D spectral data from (Panelsa, c, & e) control macrophages and (Panels b, d, & f) Δ¹-KstD expressingmacrophages incubated with 15.7 μg (10 μM) progesterone spiked with 100nCi C4-¹⁴C labeled PD (t_(r)=13.8 min; λ_(max)Of 245 nm) for 72 hours.Analysis of U-937 Δ¹-KstD cells shows formation of a new peak with aretention time of 10.0 min, a λ_(max)Of 247 nm, and containing C4-¹⁴Cscintillation events corresponding to the formation of PDD (t_(r)=10.0min, λ_(max)247 nm) following 72 hours incubation with PD. In contrast,U-937 control cells lack the ability to catabolize PD to PDD, as seen bythe absence of a peak with a 10.0 min retention time.

FIG. 60. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationby U-937-derived macrophages expressing Δ¹-KstD. Representative 2-Dchromatograms (λ280 nm) and 3-D spectral data from (Panels a & c)control U-937-derived macrophages and (Panels b & d) U-937-derivedmacrophages expressing Δ¹-KstD incubated with 17 μg (10 μM)9-hydroxypregn-4-ene-3,20-dione (9-OHPD, t_(r)=5.2 min) produced andisolated from bacterial KshAB lysate. Analysis of the U-937-derivedmacrophages expressing Δ¹-KstD following 72 hours incubation shows theformation of 3-HSP (t_(r)=7.2 min; λ_(max)280 nm). In contrast, controlU-937-derived macrophages lack the metabolic capability to produce3-HSP.

FIG. 61. Modification of the eukaryotic KshAB construct withmitochondrial targeting sequences using Gibson Assembly. The DNAencoding the Ksh A and B subunits was modified to include Aconitase2mitochondrial targeting sequences (MTS) using Gibson Assembly andsynthetic DNA. The starting vector was linearized by double restrictionenzyme digest to remove the KshA subunit and N-terminus of KshB. Thenucleotide sequence was replaced with synthetic DNA encoding KshA andthe N-terminus of KshB with the addition of a 5′ MTS attached to eachsubunit. Each repair string contained 40 bp of homology for correctincorporation into the backbone vector. The vector was reassembled usingGibson assembly as described in methods.

FIG. 62. Western blot analysis of Hep3B cells transiently expressingEF1α driven mitochondrial KshAB or cytosolic KshAB. Hep3B cells weretransiently transfected with pDest51-MTS KshAB (mitochondrial) orpDest51-KshAB (cytosolic) plasmids in 60 mm dishes and proteinexpression was assessed following 48 hours incubation. Followingincubation, cells were collected by scraping in 500 μL RIPA buffer andmechanically lysed on ice using a syringe with a 27 gauge needle.Protein samples were mixed with an equal volume of 2× Laemmli samplebuffer, boiled for 5 min, and spun at 15,000×g for 10 min at 4° C.Protein samples (25 μg) were separated using SDS-PAGE on a 10%polyacrylamide gel, transferred to a PVDF membrane, and probed withanti-FLAG (1:1000) or anti-HA (1:3000). ECL anti-mouse IgG secondaryantibody conjugated to HRP (1:10,000) and SuperSignal West FemtoSubstrate was used for detection. Samples include Hep3B cells expressingpDest51-MTS KshAB, pDest51-KshAB, and Hep3B negative control cells.

FIG. 63. RP-HPLC analysis of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)formation from progesterone (PD) utilization by the mitochondriallocalized KshAB enzyme. Representative 2-D chromatograms of (Panels a &b) λ245 nm and (Panels c & d) C4-¹⁴C scintillation events from (Panels a& c) Hep3B cells stably expressing cytosolic KshAB and (Panels b & d)Hep3B cells transiently expressing MTS-KshAB. Cells were incubated with15.7 μg (10 μM) progesterone spiked with 100 nCi C4-14C labeled PD(t_(r)=13.8 min) for 48 hour. Analysis of Hep3B cells expressingcytosolic KshAB (Panels a & c) shows a lack in ability to produce 9-OHPD(t_(r)=5.2 min, λ_(max)245 nm). In contrast, Hep3B MTS-KshAB cellsdemonstrate the ability to completely utilize the PD substrate toproduce the 9-OHPD product.

FIG. 64. Micrographs revealing addition of mitochondrial targetingsequences localizes KshAB to the mitochondria. Hep3B cells weretransfected with either (A-C) pEF-Dest51-MTS-KshAB (mitochondrial) or(D-F) pEF-Dest51-KshAB (cytosolic) for 48 hours. Cells wereimmunostained with antibody against the HA-tag of the KshB subunit(green) and co-stained with MitoTracker Far-red (purple). Mergedchannels show greater signal colocalization (white) between the KshBsubunit and Mitotracker in the pEF-Dest51-MTS-KshAB transfected cells(Pearson's coefficient=0.71) than between the KshB subunit andMitoTracker in the pEF-Dest51-KshAB (cytosolic) transfected cells(Pearson's coefficient=0.4).

FIG. 65. RP-HPLC analysis of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)formation from progesterone (PD) utilization by Hep3B MTS-KshAB cells.Representative 2-D chromatograms of (Panel a) λ245 nm and (Panel b)C4-¹⁴C scintillation events from Hep3B MTS-KshAB cells incubated with15.7 μg (10 μM) progesterone spiked with 100 nCi C4-14C labeled PD(t_(r)=13.8 min) at 1, 12, 24, 36, and 48 hour time points. Analysis ofHep3B MTS-KshAB cells reveal reduction in PD AUC and C4-¹⁴C PDscintillation events over the 48 hour time course. Concomitant to PDcatabolism, 9-OHPD (t_(r)=5.2 min; λ_(max) 245 nm) accumulates with timeas observed with a new peak forming at 5.2 min with a λ_(max)Of 245 nmand C4-¹⁴C scintillation events.

FIG. 66. Quantitative analysis of 9-hdyroxypregn-4-ene-3,20-dione(9-OHPD) product formation from progesterone (PD) catabolism by Hep3BMTS-KshAB cells. Bar graphs representing the measured (Panels a & b)area under the curve (AUC) and (Panels c & d) counts under the curve(CUC) of (Panels a & c) PD and (Panels b & d) 9-OHPD from Hep3BMTS-KshAB cells incubated with 15.7 μg (10 μM) progesterone spiked with100 nCi C4-¹⁴C labeled PD. The AUC (λ245) and CUC of PD and 9-OHPD weremeasured from 2-D chromatograms and C4-¹⁴C scintillation events underthe curve at time points: 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours.Quantitative analysis of PD and 9-OHPD AUC and CUC reveals Hep3BMTS-KshAB cells are equipped with the ability to catabolize PD to formthe hydroxylated product, 9-OHPD.

FIG. 67. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) substrate.Representative 2-D chromatogram at λ247 nm of an 80 μL injection of 15.6μg PDD in 500 μL HPLC running buffer 2. PDD was produced and isolatedfrom partially purified Δ¹-KstD enzyme incubated with progesterone (PD).The PDD was subsequently used as substrate for Hep3B MTS-KshAB cells.The PDD substrate standard in (Panel a) has a λ_(max) of 247 nm and a10.0 min retention time (t_(r)).

FIG. 68. RP-HPLC and quantitative analysis of Hep3B control cells andtheir inability to produce the ring opened product,3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP).Representative 2-D chromatogram at (Panel a) λ280 nm from Hep3B controlcells incubated with 15.6 μg (10 μM) pregn-1,4-diene-3,20-dione (PDD;t_(r)=10.0 min; λ_(max) 247 nm) from 24, 48, and 72 hour time points.Analysis of Hep3B control cells at (Panel a) λ280 nm reveals Hep3Bcontrol cells lack the ability to produce 3-HSP (t_(r)=7.2 min, λ_(max)280 nm). (Panel b) Bar graphs representing the measured area under thecurve (AUC) of 3-HSP from Hep3B control cells incubated with 7.85 μg (5μM) PDD. The AUC of 3-HSP was measured at λ280 nm from 2-D chromatogramsat time points: 24, 48, and 72 hours. Quantitative analysis of 3-HSP AUCreveals that Hep3B control cells lack 9α-hydroxylase activity, and thusare unable to produce 3-HSP.

FIG. 69. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from pregn-1,4-diene-3,20-dione (PDD) C9 hydroxylation byHep3B MTS-KshAB cells. Representative 2-D chromatograms at (Panel a)λ245 nm and (Panel b) λ280 nm from time points: 2, 12, 24, and 36 fromHep3B MTS-KshAB cells incubated with 7.85 μg (5 μM)pregn-1,4-diene-3,20-dione (PDD; t_(r)=10.2 min) produced and isolatedfrom partially purified Δ¹-KstD. Analysis of Hep3B MTS-KshAB cells at(Panel a) λ245 nm reveal reduction and exhaustion of the substrate PDDover the 36 hour time course. Concomitant to the catabolism of PDD, anew peak at (Panel b) λ280 nm, corresponding to the formation of 3-HSP(t_(r)=7.2 min, λ_(max) 280 nm) is observed with time.

FIG. 70. Quantitative analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from pregn-1,4-diene-3,20-dione (PDD) C9 hydroxylation byHep3B MTS-KshAB cells. Bar graphs representing the measured area underthe curve (AUC) of (Panel a) PDD and (Panel b) 3-HSP from Hep3BMTS-KshAB cells incubated with 7.85 μg (5 μM) PDD. The AUC of PDD and3-HSP were measured at λ245 nm and λ280 nm, respectively, from 2-Dchromatograms at time points: 2, 4, 8, 12, 24, and 36 hours.Quantitative analysis of PDD AUC reveals Hep3B MTS-KshAB cells areequipped with the ability to hydroxylate C9 of PDD to form the ringopened product, 3-HSP.

FIG. 71. RP-HPLC analysis of pregn-1,4-diene-3,20-dione (PDD) C9hydroxylation by Hep3B MTS-KshAB cells. Representative 2-D chromatogramsat (Panel a) λ245 nm and (Panel b) λ280 nm from time points: 36, 48, 60,and 72 hours from Hep3B MTS-KshAB cells incubated with 7.85 μg (5 μM)pregn-1,4-diene-3,20-dione (PDD; t_(r)=10.2 min; λ_(max)247 nm) producedand isolated from partially purified Δ¹-KstD. Analysis of Hep3BMTS-KshAB cells at (Panel a) λ245 nm shows the substrate PDD wasexhausted over the remaining 72 hour time course. In addition (Panel b)λ280 nm reveals that once the substrate PDD is exhausted, the new peakcorresponding to 3-HSP (t_(r)=7.2 min, λ_(max) 280 nm) decreases withtime. This finding suggest that Hep3B cells are equipped with additionalmetabolic capability to catabolize 3-HSP resulting in furtherdegradation of the ring opened product.

FIG. 72. Quantitative analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationand catabolism from pregn-1.4-diene-3,20-dione (PDD) C9 hydroxylation byHep3B MTS-KshAB cells. Bar graphs representing the measured area underthe curve (AUC) of (Panel a) PDD and (Panel b) 3-HSP by Hep3B MTS-KshABcells following incubation with 7.85 μg (5 μM) PDD produced and isolatedfrom partially purified Δ¹-KstD. The AUC of PDD and 3-HSP were measuredat λ245 nm and λ280 nm, respectively, from 2-D chromatograms at timepoints: 36, 48, 60, and 72 hours. Quantitative analysis of PDD AUC showsthat by 36 hours, Hep3B MTS-KshAB cells catabolized all substrate.Additionally, analysis of 3-HSP AUC reveals Hep3B MTS-KshAB cells areequipped with the ability to further catabolize the ring opened product,3-HSP.

FIG. 73. Overview of the quantitative analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationand catabolism from pregn-1.4-diene-3,20-dione (PDD) C9 hydroxylation byHep3B MTS-KshAB cells. Bar graphs representing the measured area underthe curve (AUC) of PDD and 3-HSP by Hep3B MTS-KshAB cells followingincubation with 7.85 μg (5 μM) PDD produced and isolated from partiallypurified Δ¹-KstD. The AUC of PDD and 3-HSP were measured at λ245 nm andλ280 nm, respectively, from 2-D chromatograms at time points: 2, 4, 8,12, 24, 36, 48, 60, and 72 hours. Quantitative analysis of PDD AUC showsHep3B MTS-KshAB cells hydroxylated all PDD substrate to form 3-HSP by 36hours. Analysis of 3-HSP AUC reveals Hep3B MTS-KshAB cells had maximalproduction of 3-HSP by 36 hours. In addition, it appears Hep3B cells areequipped with the ability to further catabolize the ring opened product3-HSP, as seen by the reduction in accumulated product at subsequenttime points.

FIG. 74. RP-HPLC analysis of 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)formation from progesterone (PD) utilization by MTS-KshAB expressingU-937-derived macrophages. Representative 2-D chromatograms of (Panels a& b) λ245 nm, (Panels c & d) C4-¹⁴C scintillation events, and (Panels e& f) 3-D spectral data from (Panels a, c, & e) control macrophages and(Panels b, d, & f) MTS-KshAB expressing macrophages incubated with 15.7μg (10 μM) progesterone (PD) spiked with 100 nCi C4-¹⁴C labeled PD(t_(r)=13.8 min) for 72 hours. Analysis of the MTS-KshAB expressingmacrophages reveal the PD substrate was exhausted by 72 hours.Concomitant to PD catabolism, 9-OHPD (t_(r)=5.2 min) is observed by theformation of a new peak with a retention time of 5.2 min, a λ_(max) of245 nm, and confirmed by C4-¹⁴C scintillation events. In contrast, U-937control cells lack the ability to catabolize PD to 9-OHPD, as seen bythe absence of a peak with a 5.2 min retention time.

FIG. 75. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) formationby U-937-derived macrophages expressing MTS-KshAB. Representative 2-Dchromatograms (λ280 nm) from (Panels a & c) control U-937-derivedmacrophages and (Panels b & d) U-937-derived macrophages expressingMTS-KshAB incubated with 15.6 μg (10 μM) pregn-1,4-diene-3,20-dione(PDD, t_(r)=10.0 min) produced and isolated from bacterial Δ¹-KstDlysate. Analysis of U-937 MTS-KshAB cells following 72 hours incubationshows the formation of 3-HSP (t_(r)=7.2 min). In contrast, control U-937cells lack the metabolic capability to produce 3-HSP.

FIG. 76. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from progesterone (PD) by Hep3B cells transiently expressingMTS-KshAB and Δ¹-KstD. Representative 2-D chromatograms at (Panel a)λ245 nm, (Panel b) λ280 nm, and (Panel c) C4-¹⁴C scintillation eventsfrom Hep3B cells transiently expressing EF1α driven MTS-KshAB andΔ¹-KstD. Cells were incubated with 15.7 μg (10 μM) progesterone spikedwith 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) and time points taken at6, 12, 24, and 36 hours. Analysis at (Panel a) λ245 nm reveals a largeproportion of the PD substrate being converted to9-hydroxypregn-4-ene-3,20,dione (9-OHPD, t_(r)=5.2 min) by 6 hours.Although pregn-1,4-diene-3,20-dione (PDD; t_(r)=10.0 min) is notobserved at the 6 hour time point, analysis of (Panel b) λ280 nm and(Panel c) C4-¹⁴C scintillation events reveals the formation of 3-HSP(t_(r)=7.2 min, λ_(max) 280 nm). By 12 hours, the PD substrate and9-OHPD product are exhausted resulting in maximal production of 3-HSP.Interestingly, both the area and counts under the curve of 3-HSPdecreases at further time points, suggesting that Hep3B cells haveability to further modify the pregnanering once opened. Evidence of thiscan be observed at 24 and 36 hour time points as new C4-¹⁴Cscintillation events appear between 6.0-6.5 minutes.

FIG. 77. Assembly of the MTS-KshAB-T2A-Δ¹-KstD and MTS-KshAB-P2A-Δ¹-KstDtricistronic vectors for co-expression of MTS-KshAB and Δ¹-KstD from asingle construct. The MTS-KshAB vector was linearized by restrictionenzyme digest. The DNA encoding Δ¹-KstD was obtained by doublerestriction enzyme digest. Following isolation by electrophoresis andgel extraction, Δ¹-KstD was ligated into the MTS-KshAB vector. Thepreliminary MTS-KshAB Δ¹-KstD vector was linearized by doublerestriction enzyme digest to remove the C-terminal end of KshBcontaining the native stop codon and the N-terminus of Δ¹-KstD. Tworepair strings were synthesized encoding the C-terminal end of KshB withthe native stop codon omitted, a Thosea asigna 2A skipping peptide (T2A)or a Porcine teschnovirus-1 2A skipping peptide, a Flag tag fordetection of Δ¹-KstD, and the N-terminus of Δ¹-KstD that was removed.The backbone vector and synthetic DNA were reassembled using Gibsonassembly to produce two tricistronicconstructs for the co-expression ofMTS-KshAB and Δ¹-KstD.

FIG. 78. Western blot analysis of Hep3B cells expressing EF1α drivenMTS-KshAB-P2A-Δ¹-KstD or MTS-KshAB-T2A-Δ¹-KstD constructs. Hep3B cellswere transiently transfected with pDest51-KshAB-P2A-Δ¹-KstD orpDest51-KshAB-T2A-Δ¹-KstD plasmids in 60 mm dishes and proteinexpression was assessed following 48 hours incubation. Cells werecollected by scraping in 500 μL RIPA buffer and mechanically lysed onice using a syringe with a 27 gauge needle. Protein samples were mixedwith an equal volume of 2× Laemmli sample buffer, boiled for 5 min, andspun at 15,000×g for 10 min at 4° C. Protein samples (25 μg) wereseparated using SDS-PAGE on a 10% polyacrylamide gel, transferred toPVDF membranes, and probed with anti-FLAG (1:1000) or anti-HA (1:3000).ECL anti-mouse IgG secondary antibody conjugated to HRP (1:10,000) andSuperSignal West Femto Substrate was used for detection. Samples includethe P2A construct, T2A construct, Hep3B CMV-MTS KshAB cell line(positive KshA FLAG and KshB HA control), Hep3B CMV-Δ¹-KstD cell line(positive Δ¹-KstD FLAG control), and non-transduced Hep3B cells(negative control).

FIG. 79. RP-HPLC analysis of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) productformation from progesterone (PD) catabolism by Hep3B cells expressingEF1α driven KshAB P2A Δ¹-KstD or KshAB T2A Δ¹-KstD constructs. Hep3Bcells were transiently transfected with pDest51-KshAB P2A Δ¹-KstD orpDest51-KshAB T2A Δ¹-KstD plasmids in 60 mm dishes. Following 48 hoursof protein expression, cells were incubated with 15.7 μg (10 μM) PDspiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) for 48 hours.Representative 2-D chromatograms at (Panels a & d) λ 245 nm, (Panels b &e) λ 280 nm, and (Panels c & f) C4-4C scintillation events demonstratethe efficiency of the P2A and T2A constructs in producing 3-HSP(t_(r)=7.2 min, λ_(max) 280 nm) through PD catabolism. Analysis of theP2A construct at (Panel a) λ 245 nm reveals a large proportion of the PDsubstrate being converted to 9-hydroxypregn-4-ene-3,20,dione (9-OHPD,t_(r)=5.2 min) by 48 hours. However, in comparison to the T2A constructat (Panel d) λ 245 nm, residual 9-OHPD is observed, suggesting Δ¹-KstDis the rate limiting step in 3-HSP (t_(r)=7.2 min, λ_(max) 280 nm)formation. Although 3-HSP is not observed at (Panel b) λ 280 nm or in(Panel c) C4-¹⁴C scintillation events, the accumulation of scintillationevents from an unidentified metabolite (t_(r)=2.3 min) are detectedprior to the 5.2 minute retention time of 9-OHPD. In contrast, the T2Aconstruct at (Panel d) λ 245 nm reveals complete reduction in the PDsubstrate, 9-OHPD, and pregn-1,4-diene-3,20-dione (PDD, t_(r)=10.0 min,λ_(max) 247 nm) by 48 hours. In addition, (Panel e) λ 280 nm reveals theformation of 3-HSP. Furthermore, (f) C4-¹⁴C scintillation events confirmthe formation of 3-HSP as well as additional scintillation events fromunidentified metabolites prior to 3-HSP's 7.2 minute retention time.

FIG. 80. Assembly of theP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD pentacistronicvector for co-expression of all required cholesterol catabolizingenzymes from a single construct (cholesterol catabolism cassette orCCC). The cholesterol catabolizing cassette (CCC) was assembled bylinearizing the MTS-KshAB-T2A-Δ¹-KstD vector by restriction enzymedigest. Additionally, the DNA encoding the P450-FdxR-Fdx-P2A-HSD2 wasobtained by double restriction enzyme digest. Following isolation byelectrophoresis and gel extraction, the MTS-KshAB-T2A-Δ¹-KstD backbonevector and P450-FdxR-Fdx-P2A-HSD2 fragment were assembled using tworepair strings and Gibson Assembly.

FIG. 81. Western blot analysis of Hep3B cells expressing EF1α drivenP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD pentacistronicvector (cholesterol catabolizing cassette or CCC). Hep3B cells weretransiently transfected with thepDest51-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD (CCC)plasmid in 60 mm dishes and protein expression was assessed following 48hours incubation. Cells were collected by scraping in 500 μL RIPA bufferand mechanically lysed on ice using a syringe with a 27 gauge needle.Protein samples were mixed with an equal volume of 2× Laemmli samplebuffer, boiled for 5 min, and spun at 15,000×g for 10 min at 4° C.Protein samples (25 μg) were separated using SDS-PAGE on a 10%polyacrylamide gel, transferred to PVDF membranes, and probed withanti-FLAG (1:1000) or anti-HA (1:3000). ECL anti-mouse IgG secondaryantibody conjugated to HRP (1:10,000) and SuperSignal West FemtoSubstrate were used for detection. Samples include Hep3B control cells,Hep3B CMV-MTS KshAB cells (positive KshA FLAG and KshB HA control),Hep3B CMV-Δ¹-KstD cells (positive Δ¹-KstD FLAG control), Hep3BCMV-P450-FdxR-Fdx-P2A-HSD2 cells, Hep3B EF1α-MTS-KshAB-T2A-Δ¹-KstDcells, and Hep3BEF1α-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD cells.

FIG. 82. RP-HPLC analysis of progesterone (PD) catabolism by Hep3B cellsexpressing EF1α drivenP450-FdxR-FdxP2A-HSD2-T2A-MTS-KshA-P2A-MTS-KshB-T2A-Δ¹-KstD construct(cholesterol catabolism cassette or CCC). Hep3B cells were transientlytransfected with pDest51-CCC in 60 mm dishes. Following 48 hours, cellswere incubated with 15.7 μg (10 μM) progesterone spiked with 100 nCiC4-¹⁴C labeled PD (t_(r)=13.8 min) for 24 (a, b, & c) and 72 (Panels d,e, & f) hours. Representative 2-D chromatograms at (Panels a & d) λ 245nm, (Panels b & e) λ 280 nm, and (Panels c & f) C4-¹⁴C scintillationevents demonstrate the efficiency of the CCC at catabolizing PD.Analysis of the CCC at (Panel a) λ 245 nm shows the formation ofpregn-1,4-diene-3,20-dione (PDD). Analysis of the (Panel c) C4-¹⁴Cscintillation events reveals the formation of9-hydroxypregn-4-ene-3,20-dione (9-OHPD) and confirms the production ofPDD. By 72 hours, Hep3B cells expressing the CCC catabolized a largeproportion of the PD substrate and only residual 9-OHPD, PDD, and 3-HSPwere observed. Interestingly, (f) C4-¹⁴C scintillation events were foundto accumulate in the solvent front. The scintillation events in thesolvent front are likely from an unidentified metabolite(s) (t_(r)=2-3min) that form as a result of 3-HSP metabolism by endogenous enzymes.

FIG. 83. Comparison of C4-¹⁴C-cholesterol retention between U-937control and CCC macrophages. U-937 monocytes (CCC and control) wereplated in 60 mm dishes and differentiated into macrophages (as describedin methods). Five day old macrophages were loaded with 5 μgC4-¹⁴C-cholesterol labeled LDLs for 24 hours. Following incubation,media was removed, cells were washed with PBS, and new media wasprovided. Cholesterol retention was monitored by measuring C4-¹⁴Cscintillation events in cells at timed intervals for 48 hours. Totalscintillation events per dish were normalized to cellular protein.Analysis of C4-¹⁴C scintillation events reveal the CCC cell line retainless C4-14C-cholesterol in comparison to control macrophages. Datarepresents two experiments (N=2) with four replicates each. Error barsindicate the standard error of the mean.

FIG. 84. Amino acid sequence of the P450-FdxR-Fdx fusion proteinconstruct. The minimal structures of the P450 cholesterol side chaincleavage enzyme (CYP11A1), ferredoxin reductase (FdxR), and ferredoxin(Fdx) enzymes were fused using short linkers. Green indicates the P450enzyme, red indicates the Ferrodoxin Reductase enzyme, blue indicatesthe ferrodoxin enzyme, and purple indicates mitochondrial targetingsequence. Also shown in black are the linker peptides fusing the P450sccto FdxR and FdxR to Fdx. In addition, a Flag tag (orange) and P2Apeptide (grey) were added to the 3′ end of the Fdx protein. (SEQ ID NOs:5-8)

FIG. A1. Humanized cholesterol dehydrogenase (CholD) map. The amino acidsequence of CholD from Mycobacterium tuberculosis(coding region 166-1284nt, 371 amino acids) was reverse translated using GeneOptimizer softwareset to H. sapiens codon usage. GeneOptimizer software was also used todesign flanking sequences that contained Gateway attachment sites (attB1and attB2) and restriction enzyme recognition sites (5′: MfeI and BamHI;3′: SmaI, EcoRI, and BgIII) to aid sub cloning. In addition, 5′ of theCholD sequence a TEV site, a 6× His tag, Kozak consensus sequence, atetracysteine tag, and a Flag tag were added to aid purification anddetection of the recombinant protein after expression. This humanizedCholD construct was then synthesized and inserted into the pMK-RQ vector(GeneArt).

FIG. A2. Features of the humanized cholesterol dehydrogenase (CholD)nucleotide sequence. SEQ ID NO: 10

FIG. A3. Map of humanized cholesterol dehydrogenase (CholD) in pEntr221.To generate the pEntr221-CholD entry vector, pMK-RQ-CholD and pDonr221were recombined using BP Clonase II. The resulting 3,821 nt constructincludes the humanized CholD coding sequence (653-1964 nt), 5′ TEV site,6× His Tag, Kozak consensus sequence, tetracysteine tag, and Flag tagflanked by attL attachment sites.

FIG. A4. Map of humanized cholesterol dehydrogenase (CholD) inpBAD-Dest49. To generate the pBAD-Dest49-CholD expression vector,pEntr221-CholD and pBAD-Dest49 were recombined using LR Clonasell. Theresulting 5,783 nt construct includes the humanized CholD codingsequence (727-2038 nt), 5′ TEV site, 6× His Tag, Kozak consensussequence, tetracysteine tag, and Flag tag flanked by attB attachmentsites. The pBAD-Dest49 vector expresses CholD as an N-terminal His-PatchThioredoxin fusion protein under the control of an arabinose induciblepromoter.

FIG. A5. Humanized anoxic cholesterol catabolism B enzyme (acmB) map.The amino acid sequence of acmB from Sterolibacterium denitrificans(coding region 74-1756 nt, 569 amino acids) was reverse translated usingGeneOptimizer software set to H. sapiens codon usage. GeneOptimizersoftware was also used to design flanking sequences that containedGateway attachment sites (attB1 and attB2) and restriction enzymerecognition sites (5′: MfeI and BamHI; 3′: NaeI, SmaI, EcoRI, and BgIII)to aid sub cloning. In addition, 5′ of the acmB sequence a TEV site,Kozak consensus sequence and 3′ HA tag were added to aid in purificationand detection of the recombinant protein after expression. Thishumanized CholD construct was then synthesized and inserted into thepMA-RQ vector (GeneArt).

FIG. A6. Features of the humanized anoxic cholesterol metabolism Benzyme (acmB) nucleotide sequence. SEQ ID NO: 11

FIG. A7. Map of humanized anoxic cholesterol catabolism B enzyme (acmB)in pEntr221. To generate the pEntr221-acmB entry vector, pMA-RQ-acmB andpDonr221 were recombined using BP Clonase II. The resulting 4,325 ntconstruct includes the humanized acmB coding sequence (653-2468 nt), 5′TEV site, Kozak consensus sequence and 3′ HA tag flanked by attLattachment sites.

FIG. A8. Map of humanized anoxic cholesterol catabolism B enzyme (acmB)in pBAD-Dest49. To generate the pBAD-Dest49-acmB expression vector,pEntr221-acmB and pBAD-Dest49 were recombined using LR Clonase II. Theresulting 6,287 nt construct includes the humanized acmB coding sequence(727-2542 nt), 5′ TEV site, Kozak consensus sequence and 3′ HA tagflanked by attB attachment sites. The pBAD-Dest49 vector expresses acmBas an N-terminal His-Patch Thioredoxin fusion protein under the controlof an arabinose inducible promoter.

FIG. A9. Map of humanized anoxic cholesterol catabolism B enzyme (acmB)in pLenti CMV Blast DEST (706-1). To generate the pLenti CMV Blast DEST(706-1)-acmB lentiviral expression vector, pEntr221-acmB and pLenti CMVBlast DEST (706-1) were recombined using LR Clonase II. The resulting9,458 nt construct includes the humanized acmB coding sequence(4773-6588 nt), 5′ TEV site, Kozak consensus sequence and 3′ HA tagflanked by attB attachment sites. The pLenti CMV Blast DEST (706-1)expression vector is a third generation lentiviral transfer vector thatexpresses acmB under the control of a CMV promoter and encodes forblasticidin resistance.

FIG. A11. P450-FdxR-Fdx-P2A-HSD2 construct map. The amino acid sequenceof the P450-FdxR-Fdx-P2A-HSD2 construct (coding region 122-4672 nt;P450-FdxR-Fdx: 122-3,463 nt, 1,114 amino acids; HSD2: 2554-4672 nt, 372amino acids) was reverse translated using GeneOptimizer software set toH. sapiens codon usage. GeneOptimizer software was also used to designflanking sequences that contained Gateway attachment sites (attL1 andattL2) and restriction enzyme recognition sites (5′ BgIII and XbaI; 3′BamHI and MfeI) to aid in sub cloning. The P450-FdxR-Fdx-P2A-HSD2construct was then synthesized and inserted into pMK-RQ vector(GeneArt).

FIG. A12. Features of the P450-FdxR-Fdx-P2A-HSD2 nucleotide sequence.SEQ ID NO: 12

FIG. A13. Map of P450-FdxR-Fdx-P2A-HSD2 in pEF-Dest51. To generatepEF-Dest51-P450-FdxR-Fdx-P2A-HSD2, pMK-RQ-P450-FdxR-Fdx-P2A-HSD2 andpEF-Dest51 were recombined using LR Clonase II. The resulting 10,396 ntconstruct includes the P450-FdxR-Fdx-P2A-HSD2 construct coding sequence(1752-6302 nt), 5′ Kozak consensus sequence and 3′ P450-FdxR-Fdx Flagtag flanked by attB attachment sites. The pEF-Dest51 expression vectorexpresses P450-FdxR-Fdx-P2A-HSD2 construct under the control of an EF1αpromoter and encodes for blasticidin resistance.

FIG. A14. Map of P450-FdxR-Fdx-P2A-HSD2 in pLenti CMV Blast DEST(706-1). To generate the pLenti CMV Blast DEST(706-1)-P450-FdxR-Fdx-P2A-HSD2 lentiviral expression vector,pMK-RQ-P450-FdxR-Fdx-P2A-HSD2 and pLenti CMV Blast DEST (706-1) wererecombined using LR Clonase II. The resulting 12,263 nt constructincludes the P450-FdxR-Fdx-P2A-HSD2 coding sequence (4,796-9,346 nt), 5′Kozak consensus sequence and 3′ P450-FdxR-Fdx Flag tag flanked by attBattachment sites. The pLenti CMV Blast DEST (706-1) expression vector isa third generation lentiviral transfer vector that expresses theP450-FdxR-Fdx-P2A-HSD2 construct under the control of a CMV promoter andencodes for blasticidin resistance.

FIG. A15. Humanized 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) map. Theamino acid sequence of Δ¹-KstD from Rhodococcus erythropolis (codingregion 170-1704 nt, 510 amino acids) was reverse translated usingGeneOptimizer software set to H. sapiens codon usage. GeneOptimizersoftware was used to design flanking sequences that contained Gatewayattachment sites (attB1 and attB2) and restriction enzyme recognitionsites (5′: MfeI and BamHI; 3′: EcoRI, and BgIII) to aid sub cloning. Inaddition, 5′ of the Δ¹-KstD sequence a TEV site, a 6× His tag, Kozakconsensus sequence, tetracysteine tag, and a Flag tag were added to aidin purification and detection of the recombinant protein afterexpression. This humanized Δ¹-KstD construct was then synthesized andinserted into the pUC57 vector (GenScript).

FIG. A16. Features of humanized 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD)nucleotide sequence. SEQ ID NO: 13

FIG. A17. Map of 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) in pEntr221.To generate the pEntr221-Δ¹-KstD entry vector, pUC57-Δ¹-KstD andpDonr221 were recombined using BP Clonase II. The resulting 4,247 ntconstruct includes the humanized Δ¹-KstD coding sequence (811-2390 nt),5′ TEV site, 6× His tag, Kozak consensus sequence, tetracysteine tag,and Flag tag flanked by attL attachment sites.

FIG. A18. Map of humanized 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) inpBAD-Dest49. To generate the pBAD-Dest49-Δ¹-KstD expression vector,pEntr221-Δ¹-KstD and pBAD-Dest49 were recombined using LR Clonase II.The resulting 6,209 nt construct includes the humanized Δ¹-KstD codingsequence (727-2464 nt), 5′ TEV site, 6× His tag, Kozak consensussequence, tetracysteine tag, and Flag tag flanked by attB attachmentsites. The pBAD-Dest49 vector expresses Δ¹-KstD as an N-terminalHis-Patch Thioredoxin fusion protein under the control of an arabinoseinducible promoter.

FIG. A19. Map of 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD) Kozakconsensus sequence repair string. To repair the Δ¹-KstD Kozak consensussequence, a repair string (504 nt) was designed to insert a new attB1site, Kozak consensus sequence, Flag tag, and the first 13 nt ofΔ¹-KstD's N-terminus that was removed by restriction digest. The Δ1-KstDKozak consensus sequence was repaired in the pEF-Dest51 expressionvector, and then recombined into pEntr221 for further recombinations.

FIG. A20. Features of the repair string for 3-ketosteroidΔ¹-dehydrogenase (Δ¹-KstD) Kozak consensus sequence repair. SEQ ID NO:14

FIG. A21. Map of the repaired 3-ketosteroid Δ¹-dehydrogenase (RepairedΔ¹-KstD) in pEF-Dest51. Following repair of the Δ¹-KstD Kozak consensussequence by Gibson Assembly, the resulting 7,435 nt construct includesthe repaired Δ¹-KstD (1809-3388 nt), 5′ Kozak consensus sequence, andFlag tag flanked by attB attachment sites. The pEF-Dest51 expressionvector expresses the repaired Δ¹-KstD under the control of an EF1αpromoter and encodes for blasticidin resistance.

FIG. A22. Features of the nucleotide sequence for repaired 3-ketosteroidΔ¹-dehydrogenase (Repaired Δ¹-KstD) in pEF-Dest51 SEQ ID NO: 15

FIG. A23. Map of repaired 3-ketosteroid Δ¹-dehydrogenase (RepairedΔ¹-KstD) in pEntr221. To generate the pEntr221-Repaired Δ¹-KstD entryvector, pEF-Dest51-Δ¹-KstD and pDonr221 were recombined using BP ClonaseII. The resulting 4,170 nt construct includes the Repaired Δ¹-KstDcoding sequence (658-2309 nt), 5′ Kozak consensus sequence, and Flag tagflanked by attL attachment sites.

FIG. 24. Map of repaired 3-ketosteroid Δ¹-dehydrogenase (RepairedΔ¹-KstD) in pBAD-Dest49. To generate the pBAD-Dest49-Repaired Δ¹-KstDexpression vector, pEntr221-Repaired Δ¹-KstD and pBAD-Dest49 wererecombined using LR Clonase II. The resulting 6,131 nt constructincludes the Repaired Δ¹-KstD coding sequence (807-2386 nt), 5′ Kozakconsensus sequence, and Flag tag flanked by attB attachment sites. ThepBAD-Dest49 vector expresses Repaired Δ¹-KstD as an N-terminal His-PatchThioredoxin fusion protein under the control of an arabinose induciblepromoter.

FIG. A25. Map of repaired 3-ketosteroid Δ¹-dehydrogenase (RepairedΔ¹-KstD) in pLenti CMV Blast DEST (706-1). To generate the pLenti CMVBlast DEST (706-1)-Repaired Δ¹-KstD lentiviral expression vector,pEntr221-Repaired Δ¹-KstD and pLenti CMV Blast DEST (706-1) wererecombined using LR Clonase II. The resulting 9,302 nt constructincludes the Repaired Δ¹-KstD coding sequence (4,820-6,386 nt), 5′ Kozakconsensus sequence, and Flag tag flanked by attB attachment sites. ThepLenti CMV Blast DEST (706-1) expression vector is a third generationlentiviral transfer vector that expresses Repaired Δ¹-KstD under thecontrol of a CMV promoter and encodes for blasticidin resistance.

FIG. A26. Map of repaired 3-ketosteroid Δ¹-dehydrogenase (RepairedΔ¹-KstD) in pLenti CMV Puro DEST (w118-1). To generate the pLenti CMVPuro DEST (w118-1)-Repaired Δ¹-KstD lentiviral expression vector,pEntr221-Repaired Δ¹-KstD and pLenti CMV Puro DEST (w118-1) wererecombined using LR Clonasell. The resulting 9,588 nt construct includesthe Repaired Δ¹-KstD coding sequence (4,820-6,419 nt), 5′ Kozakconsensus sequence, and Flag tag flanked by attB attachment sites. ThepLenti CMV Blast DEST (706-1) expression vector is a third generationlentiviral transfer vector that expresses Repaired Δ¹-KstD under thecontrol of a CMV promoter and encodes for puromycin resistance.

FIG. A27. Prokaryotic 3-ketosteroid 9α-hydroxylase (KshAB pro) map. Theamino acid sequence of KshAB from Rhodococcus rhodochrous(KshA codingregion 160-1338 nt, 414 amino acids; KshB coding region 1427-2485, 374amino acids) was reverse translated using GeneOptimizersoftware set toE. coli codon usage. The prokaryotic KshAB vector was designed as abicistronic construct by inserting a second shine dalgarnosequencefollowing the 3′ end of KshA. The second shine dalgarno was shifted byone nucleotide to produce a second open reading frame for coexpressionof KshB. Both subunits were designed with 5′ cell penetrating peptides(CPPs) from the HIV-TAT protein (MGYGRKKRRQRRR; SEQ ID NO: 9), shortlinker peptides (amino acids: GAS), and 6× His tags.GeneOptimizersoftware was used to design flanking sequences thatcontained Gateway attachment sites (attB1 and attB2) and restrictionenzyme recognition sites (5′ BamHI; 3′ PstIand an EcoRI between the Aand B subunits) to aid in sub cloning. The prokaryotic KshAB constructwas synthesized and inserted into pMA-RQ (GeneArt).

FIG. A28. Features of the prokaryotic 3-ketosteroid 9α-hydroxylase(KshAB pro) nucleotide sequence. SEQ ID NO: 16

FIG. A29. Map of prokaryotic 3-ketosteroid 9α-hydroxylase (KshAB pro) inpEntr221. To generate the pEntr221-KshAB (pro) entry vector,pMA-RQ-KshAB (pro) and pDonr221 were recombined using BP Clonasell. Theresulting 4,969 nt construct includes the KshAB (pro) coding sequence(761-3086 nt), 5′ KshA and KshB Shine Dalgarno sequences, cellpenetrating peptides (CPPs), and 6× His tags flanked by attL attachmentsites.

FIG. A30. Map of prokaryotic 3-ketosteroid 9α-hydroxylase (KshAB pro) inpDest14. To generate the pDest14-KshAB (pro) expression vector,pEntr221-KshAB (pro) and pDest14 were recombined using LR Clonasell. Theresulting 7,052 nt construct includes the prokaryotic KshAB codingsequence (118-2509 nt), 5′ KshA and KshB Shine Dalgarno sequences, cellpenetrating peptides (CPPs), and 6× His tags flanked by attB attachmentsites. The pDest14 vector expresses KshAB (pro) under the control of anIPTG inducible promoter.

FIG. A31. Humanized 3-ketosteroid 9α-hydroxylase (KshAB euk) map. Theamino acid sequence of KshAB from Rhodococcus rhodochrous(KshA codingregion 132-1316 nt, 406 amino acids; KshB coding region 1416-2483, 367amino acids) was reverse translated using GeneOptimizersoftware set toH. sapiens codon usage. The eukaryotic KshAB vector was designed as abicistronic construct by inserting the Porcine teschovirus-12A skippingpeptide (P2A) following the 3′ end of KshA. In addition, a Kozakconsensus sequence and Flag tag were added 5′ of KshA to aid indetection of the A subunit. Similarly, an HA tag was added 5′ of KshBfor detection of the B subunit. GeneOptimizersoftware was used to designflanking sequences that contained Gateway attachment sites (attB1 andattB2) and restriction enzyme recognition sites (5′ BgIII and XbaI; 3′BamHI and MfeI) to aid in sub cloning. The eukaryotic KshAB constructwas synthesized and inserted into pMA-RQ (GeneArt).

FIG. A32. Features of the humanized 3-ketosteroid 9α-hydroxylase (KshABeuk) nucleotide sequence. SEQ ID NO: 17

FIG. A33. Map of eukaryotic 3-ketosteroid 9α-hydroxylase (KshAB euk) inpEntr221. To generate the pEntr221-KshAB (euk) entry vector,pMA-RQ-KshAB (euk) and pDonr221 were recombined using BP Clonase II. Theresulting 4,999 nt construct includes the KshAB (euk) coding sequence(733-3084 nt), 5′ Kozak consensus sequence, KshA Flag tag, KshB HA tagand Porcine teschovirus-12A skipping peptide (P2A) flanked by attLattachment sites.

FIG. 34. Map of eukaryotic 3-ketosteroid 9α-hydroxylase (KshAB euk) inpEF-Dest51. To generate pEF-Dest51-KshAB (euk), pMA-RQ-KshAB (euk) andpEF-Dest51 were recombined using LR Clonase II. The resulting 8,265 ntconstruct includes the KshAB (euk) coding sequence (1809-4160 nt), 5′Kozak consensus sequence, KshA Flag tag, KshB HA tag and Porcineteschovirus-12A skipping peptide (P2A) flanked by attB attachment sites.The pEF-Dest51 expression vector expresses KshAB (euk) under the controlof an EF1α promoter and encodes for blasticidin resistance.

FIG. A35. Map of the first repair string encoding the Aconitase2mitochondrial targeting sequence addition to the KshA subunit. To fusethe Aconitase2 mitochondrial targeting sequence (MTS) to KshA, a repairstring (1000 nt) was designed to insert the MTS 5′ of KshA whileretaining the original attL1 site, Kozak consensus sequence, and Flagtag. The KshAB MTS addition was repaired using Gibson Assembly inpEntr221 for further propagation of the mitochondrial localized KshABinto an appropriate expression vector.

FIG. A36. Features of the nucleotide sequence for the first repairstring encoding the Aconitase2 mitochondrial targeting sequence additionto the KshA subunit. SEQ ID NO: 18

FIG. A37. Map of the second repair string encoding the Aconitase2mitochondrial targeting sequence addition to the KshB subunit. To fusethe Aconitase2 mitochondrial targeting sequence (MTS) to KshB, a repairstring (600 nt) was designed to insert the MTS 5′ of KshB whileretaining the original HA tag. The KshAB MTS addition was repaired usingGibson Assembly in pEntr221 for further propagation of the mitochondriallocalized KshAB into an appropriate expression vector.

FIG. A38. Features of the nucleotide sequence for the second repairstring encoding the Aconitase2 mitochondrial targeting sequence additionto the KshB subunit. SEQ ID NO: 19

FIG. A39. Map of the mitochondrial localized 3-ketosteroid9α-hydroxylase (MTS-KshAB) in pEntr221. Following the addition ofmitochondrial targeting sequences (MTS) to the Ksh A and B subunits byGibson Assembly, the resulting 5,197 nt construct includes the repairedMTS-KshAB construct (700-3279 nt), 5′ Kozak consensus sequence, Flag tag(KshA), and HA tag (KshB) flanked by attL attachment sites.

FIG. A40. Features of the nucleotide sequence of the mitochondriallocalized 3-ketosteroid 9α-hydroxylase (MTS-KshAB) in pEntr221. SEQ IDNO: 20

FIG. A41. Map of the mitochondrial localized 3-ketosteroid9α-hydroxylase (MTS-KshAB) in pEF-Dest51. To generate thepEF-Dest51-MTS-KshAB expression vector, pEntr221-MTS-KshAB andpEF-Dest51 were recombined using LR Clonase II. The resulting 8,463 ntconstruct includes the MTS-KshAB coding sequence (1776-4355 nt), 5′Kozak consensus sequence, Flag tag (KshA), and HA tag (KshB) flanked byattB attachment sites. The pEF-Dest51 expression vector expressesMTS-KshAB under the control of an EF1α promoter and encodes forblasticidin resistance.

FIG. A42. Map of the mitochondrial localized 3-ketosteroid9α-hydroxylase (MTS-KshAB) in pLenti CMV Blast DEST (706-1). To generatethe pLenti CMV Blast DEST (706-1)-MTS-KshAB lentiviral expressionvector, pEntr221-MTS-KshAB and pLenti CMV Blast DEST (706-1) wererecombined using LR Clonase II. The resulting 10,330 nt constructincludes the MTS-KshAB coding sequence (4820-7399 nt), 5′ Kozakconsensus sequence, Flag tag (KshA), and HA tag (KshB) flanked by attBattachment sites. The pLenti CMV Blast DEST (706-1) expression vector isa third generation lentiviral transfer vector that expresses MTS-KshABunder the control of a CMV promoter and encodes for blasticidinresistance.

FIG. A43. Map of the T2A repair string encoding the Thosea asigna 2Askipping peptide for the MTS-KshAB and Δ¹-KstD tricistronic vector. Toco-express MTS-KshAB and Δ¹-KstD from a single vector, a repair string(656 nt) was designed to replace the native KshB stop codon with aThosea asigna 2A skipping peptide within the MTS-KshAB Δ¹-KstD ligatedintermediate product. The T2A repair string was designed to encode forthe C-terminal end of KshB, the T2A skipping peptide, the Δ¹-KstD Flagtag, and the N-terminus of Δ¹-KstD that was removed followingrestriction digest. The repair string included 40 bp homology armsstarting from the 3′ overhangs generated from the BbvCI and Speldigest.The MTS-KshAB was repaired using Gibson Assembly in pEntr221 for furtherpropagation of the MTS-KshAB T2A Δ¹-KstD tricistronic vector into anappropriate expression vector.

FIG. A44. Features of the nucleotide sequence of the T2A repair stringencoding the Thosea asigna 2A skipping peptide for the MTS-KshAB andΔ¹-KstD tricistronic vector. SEQ ID NO: 21

FIG. A45. Map of the P2A repair string encoding the Porcineteschovirus-1 2A skipping peptide for the MTS-KshAB and Δ¹-KstDtricistronic vector. To co-express MTS-KshAB and Δ¹-KstD from a singlevector, a repair string (659 nt) was designed to replace the native KshBstop codon with a Porcine teschovirus-12A skipping peptide in theMTS-KshAB Δ¹-KstD ligated intermediate product. The P2A repair stringwas designed to encode for the C-terminal end of KshB, the P2A skippingpeptide, the Δ¹-KstD Flag tag, and the N-terminus of Δ¹-KstD that wasremoved following restriction digest. The repair string included 40 bphomology arms starting from the 3′ overhangs generated from the BbvCIand SpeI digest. The MTS-KshAB was repaired using Gibson Assembly inpEntr221 for further propagation of the MTS-KshAB P2A Δ¹-KstDtricistronic vector into an appropriate expression vector.

FIG. A46. Features of the nucleotide sequence of the P2A repair stringencoding the Porcine teschovirus-1 2A skipping peptide for the MTS-KshABand Δ¹-KstD tricistronic vector. SEQ ID NO: 22

FIG. A47. Map of the MTS-KshAB-T2A-Δ1-KstD tricistronic construct inpEntr221. Following replacement of the native KshB stop codon with aThosea asigna 2A skipping peptide (T2A) using Gibson Assembly, theresulting 6,803 nt construct includes the repaired MTS-KshAB enzyme(1,007-3,586 nt), the Δ¹-KstD enzyme (3,674-5,212 nt), 5′ Kozakconsensus sequence, KshA Flag tag, KshB HA tag, and Δ¹-KstD Flag tagflanked by attL attachment sites. The MTS-KshAB Δ¹-KstD ligatedintermediate product was repaired in pEntr221 for further propagation ofthe MTS-KshAB-T2A-Δ¹-KstD tricistronic construct into an appropriateexpression vector.

FIG. A48. Features of the nucleotide sequence for theMTS-KshAB-T2A-Δ¹-KstD construct in pEntr221. SEQ ID NO: 23

FIG. A49. Map of the MTS-KshAB-T2A-Δ¹-KstD tricistronic construct inpEF-Dest51. To generate the pEF-Dest51-MTS-KshAB-T2A-Δ¹-KstD expressionvector, pEntr221-MTS-KshAB-T2A-Δ¹-KstD and pEF-Dest51 were recombinedusing LR Clonase II. The resulting 10,069 nt construct includes theMTS-KshAB coding sequence (1,776-4,355 nt), Δ¹-KstD (4,443-5,981 nt), 5′Kozak consensus sequence, KshA Flag tag, KshB HA tag, and Δ¹-KstD Flagtag flanked by attB attachment sites. The pEF-Dest51 expression vectorexpresses MTS-KshAB-T2A-Δ¹-KstD under the control of an EF1α promoterand encodes for blasticidin resistance.

FIG. A50. Map of MTS-KshAB-T2A-Δ¹-KstD in pLenti CMV Puro DEST (w118-1).To generate the pLenti CMV Puro DEST (w118-1)-MTS-KshAB-T2A-Δ¹-KstDlentiviral expression vector, pEntr221-MTS-KshAB-T2A-Δ¹-KstD and pLentiCMV Puro DEST (w118-1) were recombined using LR Clonase II. Theresulting 12,222 nt construct includes the MTS-KshAB coding sequence(4820-7399 nt), Δ¹-KstD (7487-9025 nt), 5′ Kozak consensus sequence,KshA Flag tag, KshB HA tag, and Δ¹-KstD Flag tag flanked by attBattachment sites. The pLenti CMV Puro DEST (w118-1) expression vector isa third generation lentiviral transfer vector that expressesMTS-KshAB-T2A-Δ¹-KstD under the control of a CMV promoter and encodesfor puromycin resistance.

FIG. A51. Map of the first repair string for insertion of theP450-FdxR-Fdx-P2A-HSD2 construct into the MTS-KshAB-T2A-Δ¹-KstDtricistronic vector. To co-express the P450-FdxR-Fdx and HSD2 enzymesalong with MTS-KshAB and Δ¹-KstD, one of two repair strings (474 nt) wasdesigned to encode for the N-terminal segment of the P450 enzyme thatwas lost following restriction digest. The repair string included 40 bpof homology starting from the 3′ overhangs of the NcoI and ScaIrestriction sites. The P450-FdxR-Fdx-P2A-HSD2 addition to theMTS-KshAB-T2A-Δ¹-KstD tricistronic vector was repaired using GibsonAssembly in pEntr221 for further propagation of the pentacistronicconstruct into an appropriate expression vector.

FIG. A52. Features of the nucleotide sequence of the first repair stringfor insertion of the P450-FdxR-Fdx-P2A-HSD2 construct into theMTS-KshAB-T2A-Δ¹-KstD tricistronic vector. SEQ ID NO: 24

FIG. A53. Map of the P450-FdxR-Fdx-P2A-HSD2 fragment for insertion intothe MTS-KshAB-T2A-Δ¹-KstD tricistronic vector. To co-express theP450-FdxR-Fdx and HSD2 enzymes along with MTS-KshAB and Δ¹-KstD, theP450-FdxR-Fdx-P2A-HSD2 fragment (3221 nt) was generated by ScaI andEcoRV restriction digest of pMK-RQ-P450-FdxR-Fdx-P2A-HSD2. TheP450-FdxR-Fdx-P2A-HSD2 addition to the MTS-KshAB-T2A-Δ¹-KstDtricistronic vector was repaired using Gibson Assembly in pEntr221 forfurther propagation of the pentacistronic construct into an appropriateexpression vector.

FIG. A54. Features of the nucleotide sequence of theP450-FdxR-Fdx-P2A-HSD2 fragment for insertion into theMTS-KshAB-T2A-Δ¹-KstD tricistronic vector. SEQ ID NO: 25

FIG. A55. Map of the second repair string for insertion ofP450-FdxR-Fdx-P2A-HSD2 construct into the MTS-KshAB-T2A-Δ1-KstDtricistronic vector. To co-express the P450-FdxR-Fdx and HSD2 enzymesalong with MTS-KshAB and Δ1-KstD, a second repair string (1079 nt) wasdesigned to encode for the C-terminal segment of the HSD2 enzyme thatwas lost following restriction digest and a Thosea asigna 2A skippingpeptide (T2A). The repair string included 40 bp of homology startingfrom the 3′ overhangs of the EcoRV and NcoI restriction enzymerecognition sites. The P450-FdxR-Fdx-2A-HSD2 addition to theMTS-KshAB-T2A-Δ1-KstD tricistronic vector was repaired using GibsonAssembly in pEntr221 for further propagation of the pentacistronicconstruct into an appropriate expression vector.

FIG. A56. Features of the nucleotide sequence of the second repairstring for insertion of the P450-FdxR-Fdx-P2A-HSD2 fragment forinsertion into the MTS-KshAB-T2A-Δ¹-KstD tricistronic vector. SEQ ID NO:26

FIG. A57. Map of theP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD pentacistronicconstruct (the cholesterol catabolizing cassette (the CCC)) in pEntr221.Following insertion of the P450-FdxR-FdxP2A-HSD2 construct into theMTS-KshAB-T2A-Δ¹-KstD tricistronic vector using Gibson Assembly, theresulting 11,414 nt construct includes the P450-FdxR-Fdx fusion protein(1007-4,348 nt), HSD2 (4,439-5,554 nt), MTS-KshAB (5747-8197 nt),Δ¹-KstD (8285-9814 nt), 5′ Kozak consensus sequence, P450-FdxR-Fdx Flagtag, KshA Flag tag, KshB HA tag, and Δ¹-KstD Flag tag flanked by attLattachment sites. The CCC was assembled in pEntr221 for furtherpropagation of the pentacistronic construct into an appropriateexpression vector.

FIG. A58. Features of the nucleotide sequence of theP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD pentacistronicconstruct (the cholesterol catabolizing cassette (the CCC)) in pEntr221.SEQ ID NO: 27

FIG. A59. Map of theP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD pentacistronicconstruct (the cholesterol catabolizing cassette (the CCC)) inpEF-Dest51. To generate thepEF-Dest51-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstDexpression vector,pEntr221-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD andpEF-Dest51 were recombined using LR Clonase II. The resulting 14,680 ntconstruct includes the P450-FdxR-Fdx fusion protein (1,776-5,117 nt),HSD2 (5,208-6,323 nt), MTS-KshAB (6,516-8,966 nt), Δ¹-KstD (9,054-10,583nt), 5′ Kozak consensus sequence, P450-FdxR-Fdx Flag tag, KshA Flag tag,KshB HA tag, and Δ¹-KstD Flag tag flanked by attB attachment sites. ThepEF-Dest51 expression vector expresses theP450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD construct under thecontrol of an EF1α promoter and encodes for blasticidin resistance.

FIG. A60. Map of P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstDpentacistronic construct (the cholesterol catabolizing cassette (theCCC)) in pLenti CMV Puro DEST (w118-1). To generate the pLenti CMV PuroDEST (w118-1)-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstDlentiviral expression vector,pEntr221-P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD and pLentiCMV Puro DEST (w118-1) were recombined using LR Clonase II. Theresulting 16,833 nt construct includes the P450-FdxR-Fdx fusion protein(4,820-8,161 nt), HSD2 (8,252-9,367 nt), MTS-KshAB (9,560-12,010 nt),Δ¹-KstD (12,098-13,627 nt), 5′ Kozak consensus sequence, P450-FdxR-FdxFlag tag, KshA Flag tag, KshB HA tag, and Δ¹-KstD Flag tag flanked byattB attachment sites. The pLenti CMV Puro DEST (w118-1) expressionvector is a third generation lentiviral transfer vector that expressesMTS-KshAB under the control of a CMV promoter and encodes for puromycinresistance.

DETAILED DESCRIPTION

Disclosed herein is the development of a unique, cell-based approach tohelp manage homozygous familial hypercholesterolemia (FH). FH patientslack functional LDL receptors, which prevents the uptake of low densitylipoproteins (LDLs) by the liver and other tissues. As a result, highlevels of circulating LDLs are presented to macrophages, which expressscavenger receptors (SRs) that take in LDLs. Because humans lack enzymesto degrade cholesterol and SRs are not sterol responsive, themacrophages fill with cholesterol and cholesterol esters (CE) and becomefoam cells. This process elicits a maladaptive immune response thatplace FH patients at extreme risk for having a heart attack, whichusually occurs within the first two decades of life. At a biochemicallevel, cholesterol accumulates because human cells do not expressenzymes that can open the cholestane ring. Applicants show, herein thatengineering human macrophages to express cholesterol ring openingenzymes can enable cholesterol catabolism in human cells. To develop thedisclosed systems, methods, and compositions, Applicants developed andcharacterized six enzymes involved in cholesterol catabolism(cholesterol dehydrogenase (CholD), 3-ketosteroid Δ¹-dehydrogenase(Δ¹-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid9α-hydroxylase (KshAB), 303-hydroxysteroid dehydrogenase 2 (HSD2), and aP450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx)).First, the bacterial enzymes were humanized and expressed in E. coli.For this effort, biochemical assays were developed to measure activityand RP-HPLC methods were developed to detect novel catabolites.Confirmation of the predicted intermediates was achieved using LiquidChromatography-Mass Spectrometry (LC-MS). Next, lentiviral expressionconstructs were developed to produce stable Hep3B and U-937 cell linesthat independently expressed each enzyme. Initially, KshAB was notactive in human cells. This obstacle was resolved by directingexpression to the mitochondria. Four enzymes were found to be sufficientto initiate cholesterol ring opening in Hep3B and U-937 cells, and bothcell lines appear to have endogenous metabolic capability allowingfurther degradation after the cholestane ring has been opened. Once theminimal number of enzymes required to initiate cholesterol degradationwas determined, human U-937 derived macrophages were engineered toexpress the required four enzymes from a 10 kb pentacistronic expressionsystem that that takes advantage of multiple viral 2A skipping peptides.Macrophages engineered to express this cholesterol catabolizingcassette, or CCC, were found to retain less cholesterol following LDLloading as compared to control macrophages, and may thus have a higherresistance to foam cell formation.

Atherosclerosis is a chronic maladaptive inflammatory response initiatedby the retention of cholesterol rich apoprotein-B containinglipoproteins within the arterial wall. Atherosclerosis is an underlyingcause of cardiovascular disease (CVD), myocardial infarction, stroke andperipheral vascular disease, which are leading causes of death in theUnited States. Inherited defects in many different aspects oflipoprotein metabolism (FIG. 3), poor diet, a sedentary lifestyle, andsecondary effects of other disorders (notably diabetes, hypothyroidismand kidney disease) all contribute to onset and progression ofatherosclerosis.

For the medical management of CVD, physicians currently have manyoptions (statins, niacin, bile acid binding resins, inhibitors ofintestinal cholesterol absorption, fibrates, fish oils etc.). Becausedefects in any one of the many different proteins that control normallipid metabolism can contribute to the progression of CVD, there is nota single treatment option that is useful for all people.

Lipid transport systems in mammals move energy rich lipids [i.e.triglycerides (TG), cholesterol, and phospholipids] from the site ofintestinal absorption and hepatic synthesis through the vascular spaceto sites of cellular utilization (FIG. 3). As depicted in FIG. 3 at (1)Dietary fat is incorporated into the core of chylomicrons, which aretransported to the liver and taken up by apoB/LDL receptor-mediatedendocytosis. In the liver, cholesterol is used to make bile salts.Cholesterol, bile salts and phospholipids are then secreted back intothe intestine as bile. FIG. 3 istem (2) depicts when the body has acaloric surplus, the liver “repackages” dietary TG and newly synthesizedTG (made from excess carbohydrate) into very low-density lipoproteins(VLDLs). Like the chylomicrons, the principle job of VLDLs is totransport TG in the blood. With the aid of lipoprotein lipase, TG inchylomicrons and VLDLs is broken down to fatty acids, which are absorbedby adipose tissue and converted back into TG for storage. Fatty acidsare also absorbed by muscle and used to produce energy viabeta-oxidation. At (3) is showing reverse cholesterol transport, whereHDLs scavenge excess cholesterol, which is converted to cholesterolesters via the actions of lecithin cholesterol acyl-transferase (LCAT).Cholesterol ester transport proteins (CETPs) aid the movement of CEsfrom HDLs to VLDL remnants (IDLs). Lipoproteins containing ApoE(remnants and HDLs) are rapidly absorbed by the liver. As VLDLs losetheir TG, they eventually become cholesterol ester rich LDLs. LDLscontain ApoB100, and can be cleared by LDL-receptor mediatedendocytosis, which occurs mainly in the liver. Apoproteins important forthe metabolism of the major lipoproteins known to be mutated in peoplewith disease are listed below the particle (e.g. E, B-100). Geneticdefects in nearly 50 ancillary proteins involved in lipid metabolism arelikely to contribute to the atherosclerotic process.

Currently most medical treatments alter some aspect of normallipoprotein metabolism to prevent the accumulation of lipoproteins(principally LDLs) in the arterial wall. Inhibitors of HMG-CoA reductase(i.e. statins) lead to an increase in LDL-receptor expression, and alarge number of clinical trials indicate that statins reduce events(e.g. heart attacks and strokes) by ˜25-35%. While this represents aremarkable achievement, a 35% reduction means 65% of the people with CVDstill have events. Stronger statins may be developed, and combinationtherapy will likely further reduce the number of events. However,complete suppression of cholesterol synthesis is not a therapeuticoption because cholesterol is important for normal biology. Humans needcholesterol as a precursor for the production of bile salts, and sterolhormones. In addition, cholesterol is a regulator of membrane fluidityin animals, so its production should not be eliminated entirely.

FIG. 1 depicts the development of CVD. In the early stages ofatherosclerosis, apoB containing lipoproteins (principally LDLs) areretained in the sub-endothelial extracellular matrix of the arterialwall (1^(st) diagram). Accumulation of LDLs in the intima initiates amaladaptive inflammatory response, marked by monocyte sub-endothelialinfiltration and differentiation into macrophages that ingest thelipoproteins. As the macrophages “fill up” with lipids, they become foamcells (2^(nd)-3^(rd) diagram). In advanced stages of CVD, plaques fillwith inflammatory cells and lipids from dead and dying macrophages. Thecontinual presence of dyslipidemia induces smooth muscle cells (SMC) tomigrate into the intima to form a fibrous cap in an attempt to “walloff” the site of inflammation (4^(th) diagram). Connective tissue islaid down, followed by calcification (hardening of the arteries). Thefibrous cap can erode and eventually rupture, inducing acute thromboticvascular events, commonly myocardial infarctions and strokes.

At a basic level, CVD is a disease of the intima. Atherogenesis startswith endothelial damage or dysfunction in the arteries, which allows theaccumulation of apoB-containing lipoproteins in the intima. Thehalf-life of LDLs in the blood is increased when the amount ofcirculating apoB-containing lipoproteins (principally LDLs) exceeds therate of hepatic clearance. This aids LDL accumulation in thesub-endothelial space of arterial walls. To clear the intima oflipoproteins and lipoprotein-debris, monocytes enter the sub-endothelialspace via a complex process (diapedesis) and differentiate intomononuclear phagocytes (macrophages). Macrophages ingest thecholesterol/CE-rich lipoproteins via many processes, includingLDL-receptor (LDL-R) and scavenger-receptor (SR)-mediated endocytosis.As more lipoproteins enter the intima, the macrophages continue toingest them. As a result, intracellular cholesterol starts toaccumulate. To protect the cell from the membrane disruptive affectsproduced by excess cholesterol, acyl-CoA acyltransferase (ACAT1) isactivated. ACAT1 converts cholesterol into cholesterol esters (CEs),which accumulate as less toxic cytoplasmic inclusions. Excesscholesterol also increases the expression of ATP-bindingcassette-transport proteins (i.e. ABCA1 and ABCG1), allowing cholesterolefflux to apoA1 and existing HDLs. This increases reverse cholesteroltransport to the liver. Intracellular cholesterol also inhibits LDL-Rexpression and triggers the degradation of existing LDL-Rs, preventingfurther uptake. However, LDL-uptake also occurs via SR-mediatedmechanisms (e.g. SR-Δ¹, SR-Bland CD36), which continues because theSR-pathways in macrophages are not suppressed by an excess of sterols.Therefore, macrophages continue to ingest LDLs that enter thesub-endothelial space. Unless HDL levels are high, uptake is moreefficient than efflux. With time the macrophages become engorged withCEs, which accumulate in intracellular droplets producing a “foamy”appearance when examined microscopically, hence the name foam cells.During this process a complex response is triggered, which elicits the“maladaptive inflammatory response” that ultimately leads to CVD.Therefore, at a fundamental level, the inability of macrophages todegrade cholesterol appears to initiate disease. Applicants hypothesizedthat if cholesterol could be degraded by macrophages, like fatty acidsand phospholipids, they would not fill up with CEs and elicit themaladaptive immune response. Thus, by enabling cholesterol catabolismthe disclosed methods and compositions may ameliorate this fundamentalaspect of disease. Because CVD is the leading cause of human death, anovel method to prevent or reduce the size of existing plaques may havea huge impact on society.

The present disclosure is based on an unexpected observation.Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis.During the chronic stage of infection, M. tuberculosis residesintracellularly in macrophages, which allows the bacteria to avoid manyhost immune responses. When unable to eradicate infection, the hostimmune system encases the infected macrophages into dense granulomasstructures. This restricts the growth of intracellular pathogens, inpart, by depriving them of essential nutrients. How mycobacteria survivein phagosomes for extended periods of time was a key unanswered questionin the field, until a surprising observation revealed M. tuberculosiscan utilize host cholesterol as a source for carbon and energy. Whensequestered into phagosomes, M. tuberculosis activate operons encodingmany genes, some of which encode proteins that enable cholesterolcatabolism. Further investigations into the molecular mechanisms of M.tuberculosis survival on host cholesterol identified two key enzymes[KstD and KshA/B] which catalyze reactions needed to open the cholestanering. Mammals do not have orthologues for either enzyme. However, bothwere active when expressed in E. Coli.

Identification of key cholesterol catabolizing enzymes. The enzymesinvolved in bacterial cholesterol catabolism have been identified andcharacterized. Catabolism occurs via two independent pathways; the C17chain degradation pathway, and the four-ring carbon skeleton degradationpathway (FIG. 2B). In humans cholesterol catabolism is limited. Asdiscussed above ACATs convert cholesterol into cholesterol esters, whichare readily converted back to cholesterol and fatty acids byCE-esterases. In steroidogenic cells the C-17 side chain is degraded bycytochrome p450s (e.g. CYP11Δ¹). During both steroid hormone and bilesalt synthesis the cholestane ring can be modified in many ways by avariety of p450s, hydroxylases, reductases and dehydrogenases. Studieswith squalene synthase inhibitors have also revealed a number ofpreviously unrecognized pathways that are capable of degrading themajority of the synthetic intermediates produced during cholesterolsynthesis and metabolism. Still, after the ring is closed, humans do nothave enzymes capable of opening the cholestane ring.

Humanization, Expression and Characterization of CholesterolDehydrogenase (CD) KstD and KshA/B.

Comparison of cholesterol metabolic and catabolic pathways revealedthree bacterial enzymes that have no orthologues in animals: 1)cholesterol dehydrogenase (CD); 2) 3-ketosteroid-Δ1-dehydrogenase(KstD), and 3) 3-ketosteroid-9α-hydroxylase (KshA/B)]. The reactionscatalyzed by these enzymes are shown in FIG. 3 (indicated as 1, 2 and 3,respectively). Together they catalyze B-ring opening and aromatizationof ring A to produce3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA).

To test the disclosed hypothesis, CD, KstD, and KshA/B were humanized.Humanized enzymes were designed in silico, produced synthetically as˜200 to 2500 base pair “strings” and assembled using Gibson Assembly.Humanization included: 1) codon optimization, 2) G/C content adaptation,3) adding components needed for eukaryotic expression (e.g. Kozakconsensus sequence, etc.) 4) elimination of cryptic splice sites and RNAdestabilizing sequence elements for increased RNA stability, and 5)avoidance of sequences that would yield stable RNA secondary structures.Other modifications were introduced to facilitate cloning, expressionand detection. Following Gibson assembly, the constructs were subclonedinto traditional expression vectors (e.g. pDest51(EF1alpha);plenti-CMV-Blast, etc.) using Gateway mediated recombination andconventional methods. For initial studies, the constructs were clonedinto expression vectors driven by conventional prokaryotic or eukaryoticpromoters. All constructs were sequenced in their entirety to ensurefidelity.

Expression of the disclosed humanized sequences may be through one ormore control sequences. In various embodiments, the control sequencesmay be selected from transcriptional enhancers, promoters, and the likethat allow for binding of an RNA polymerase. In most embodiments, theenhancer, promoter, or combinations thereof are eukaryotic promoters andenhancers and the polymerase is a Pol II polymerase. In mostembodiments, the eukaryotic promoter or enhancer is a promoter orenhancer from a virus, plant, animal, mammal, mouse, human, fungus,yeast, or insect. In some embodiments the promoter or enhancer isselected from one or more of CMV, SV40, EF1α, PGK, Ubc, and otherpromoters and enhancers known to those of skill in the art.

Humanized prokaryotic nucleotide sequences of the present disclosure maybe greater than about 60% identical, over at least about 200nucleotides, to the prokaryotic sequence. In many embodiments, thedisclosed humanized protein sequence may be greater than about 80%identical, over at least about 30 amino acids, to the prokaryotic aminoacid sequence. In many embodiments, these humanized sequences may begreater than 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and lessthan about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%,88%, 87%, 86%, 85%, 80%, 75%, 70%, or 65% identical to the prokaryoticsequences. In many embodiments the length of identity may be more than30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500,600, 700, 800, 900, or 1000 amino acids or nucleotides, and less thanabout 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 180, 160, 140,120, 100, 90, 80, 70, 60, 50, or 40 amino acids or nucleotides.

Characterization of humanized enzymes expressed in E. coli.

When expressed in E. coli, hCD was active. However, hKstD designed basedon Mycobacterial KstD showed almost no activity against cholestenone(the product produced by CD). This obstacle was resolved by theidentification, humanization and expression of another3-ketosteroid-Δ1-dehydrogenase identified in Streptomyces denitrificans(ACMB), which can utilize cholestenone as a substrate. The expression ofhKstD and hKshA/B in E. coli produced active enzyme.

Expression of the cholesterol catabolizing enzymes in human cells. Nextactivity was tested when the enzymes were expressed in human Hep3Bcells. hCD was active, but the activity of both hKstD and hACMB wasminimal. hKshA/B was completely inactive, and without furthercatabolism, the build-up of cholestenone (produced by hCD alone) killedthe cells. To overcome these obstacles, focus was placed onunderstanding why the recombinant humanized enzymes were active whenexpressed in bacteria but inactive when expressed in human cells. Thefirst insight came from the co-crystal structure of bacterial KstD incomplex with ADD (FIG. 2D).

The structures revealed that the catalytic site of KstD is a deeppocket, with two isoleucine residues at the entrance that produce asteric clash if the C-17 side chain of cholesterol has not been removed.Although there are ongoing discussions in the literature about the needto remove the side chain prior to ring opening, the co-crystalstructures provide robust evidence that the side chain is removed beforehKstD can act upon the ring. Although the bacterial side chaindegradation pathway produces ADD (ketone at C-17; FIG. 2B), the modelpredicted that 4-pregn-4-ene-3,20 dione [PD; has 2 additional side-chaincarbons] could still fit in the active site. When tested experimentally,PD was efficiently catabolized by the recombinant humanized enzymes(FIG. 2E).

When hKstD was expressed in human cells (Hep3B) it was active againstPD.

Expression of Active hKshA/B in Human Cells.

The next challenge was to determine why KshA/B was not functional.KshA/B is comprised of Rieske type non-heme monooxygenase comprised ofan oxygenase (KshA) and a reductase KshB. Fortuitously, the crystalstructure a Rhodococcus ortholog was solved by Capyk, J K et al.

Based in the crystal structure it became clear that KshA/B was likely asix polypeptide multi-protein complex. Therefore, to allow the complexto properly assemble it was critical to develop an expression systemthat would ensure an equal number of both subunits would be producedsimultaneously and in close proximity.

Nature again provided insights needed for the expression of the activehKshA/B complex in human cells. To express equal amounts of hKshA andhKshB an expression vector was constructed with both genes in frame andseparated by Porcine teschovirus 2A peptide (FIG. 2H). The Porcineteschovirus-1 has a single-stranded non-segmented RNA genome. It wasinitially believed that translation of the viral RNA produced a singlepolyprotein that was later proteolytically cleaved into 12 proteins.However, more recent studies revealed that the “2A” peptide expressedbetween proteins is not produced as a protease recognition spacer.Rather the 2A peptide has a unique action in the ribosome. 2A ends with3 key amino acids (PGP). During translation, after the prolyl-tRNA ispositioned within the peptidyltransferase center with the remainder ofthe 2A peptide in the exit tunnel, conformational restraints prevent theincorporation of the last proline into the nacent chain and promotesrelease of the growing polypeptide. Since the tRNA-(Pro) is alreadypositioned within the ribosome before the growing polypeptide isreleased, translation of the next open reading frame proceeds with veryhigh efficiency (FIG. 2H).

Enabling Cholesterol Catabolism in Cultured Human Cells—Co-Expression ofΔ¹-KstD and MTS-KshAB in Hep3B Cells

To determine whether Hep3B cells could co-express Δ¹-KstD and MTS-KshABto catalyze the formation of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) fromprogesterone (PD), Hep3B cells were transiently transfected with equalquantities of pDest51-Δ¹-KstD and MTS-KshAB. Following 48 hours foradequate protein expression, cells were incubated with 15.7 μg (10 μM)progesterone spiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) foran additional 36 hours. RP-HPLC analysis of the 36 hour time courserevealed a robust conversion of the PD substrate to 9-OHPD (λ_(max) 245nm; t_(r)=5.2 minutes) by 6 hours. Although PDD (λ_(max) 247 nm;t_(r)=10.0 minutes) was not observed at 6 hours, analysis at λ 280 nmreveals the formation of 3-HSP (FIG. 2I)

By 12 hours, the substrate and all intermediates had been catabolized toform 3-HSP. Interestingly, time points at 24 and 36 hours reveal thatafter the substrates and intermediates had been completely exhausted,3-HSP is being further metabolized. The accumulation of C4-¹⁴Cscintillation events at 6.5 minutes confirms that once the cholestanering has been opened it is being modified to have an increased polarity,as observed by a decrease in retention time.

Co-Expression of MTS-KshAB and Δ¹-KstD from a Tricistronic Vector

Following confirmation that MTS-KshAB and Δ¹-KstD could besimultaneously expressed to produce3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) from thesubstrate progesterone (PD), our next step was to design and assemble atricistronic vector for co-expressing MTS-KshAB and Δ¹-KstD from asingle construct. Two vectors were designed to co-express MTS-KshAB andΔ¹-KstD by inserting a Thosea asigna 2A skipping peptide (T2A) or aPorcine teschnovirus-1 2A skipping peptide (P2A) between the twoenzymes. The T2A and P2A vectors were characterized by assessing KshABand Δ¹-KstD protein levels by Western blot and activity by RP-HPLCanalysis. Hep3B cells were transiently transfected with the T2A and P2Avectors. Following 48 hours to allow for adequate protein expression,cells were analyzed by Western blot and duplicate dishes were incubatedwith 15.7 μg (10 μM) progesterone spiked with 100 nCi C4-¹⁴C labeled PD(t_(r)=13.8 min) for an additional 48 hours. The Western blot resultsreveal that co-expressing MTS-KshAB and Δ¹-KstD with the T2A skippingpeptide resulted in higher expression of the Flag tagged KshA subunit,the HA tagged KshB subunit, and the Flag tagged Δ¹-KstD enzyme (Example13).

In addition, RP-HPLC analysis revealed the T2A transfected cells hadhigher enzyme activity than the P2A transfected cells. The T2Atransfected cells were able to completely catabolize the PD substrateand intermediates (FIG. 79 panel d) resulting in the formation of 3-HSP(t_(r)=7.2 min, λ_(max) 280 nm) (FIG. 79 panel e) and additionaldownstream degradation products (79 panel f). The activity of the P2Aconstruct was less, as seen by the presence of residual progesterone,9-OHPD (FIG. 79 panel a), and a lack of 3-HSP formation (79 panel b &c). These findings reveal that the activity of Δ¹-KstD is the ratelimiting step in 3-HSP formation in the P2A construct.

With time 3-HSP is completely degraded, apparently by existingendogenous enzymes (i.e. 3-HSP added to non-transformed Hep3B cells isdegraded). Production of downstream metabolites does not appear toaffect the cultured cells.

Cholesterol is a necessary membrane lipid that is found in every cell ofthe body (Brown & Goldstein, 1999). Cholesterol is a planar, tetracyclicmolecule consisting of a hydrophilic hydroxyl group at C3 and ahydrophobic alkyl side chain at C17. The amphipathic nature ofcholesterol allows it to partition into phospholipid membranes where itacts as an important structural component that is essential formaintaining the fluidity and permeability of all animal membranes(Sinensky, 1978). A cell membrane that lacks adequate levels ofcholesterol becomes highly fluid, eventually leading to lysis of thecell (Anderson, 2003; Kellner-Weibel et al., 1999; Brown & Goldstein,1999; Sinensky, 1978). In contrast, excess membrane cholesteroldecreases fluidity, adversely affecting membrane permeability (Brown &Goldstein, 1999; Cooper, 1977). As such, it is critical that all cellstightly regulate the level of free cholesterol in order to maintaincholesterol homeostasis. In addition to its contribution to theproperties of membranes, cholesterol is used as a precursor for bilesalts, and steroid hormones (Russell, 1992; Russell, 2009).

De Novo Synthesis of Cholesterol

Most, if not all cells have the ability to synthesize cholesterol.Cholesterol synthesis is a complex and an energy-expensive process,requiring the coordinated activity of more than fifteen enzymes. Thebackbone of cholesterol consists of 27 carbon atoms that are assembledby multiple enzymes, with all carbons coming from acetyl-CoA andmolecular O₂ to generate the C-3 hydroxyl (FIG. 2). The acetyl-CoA isderived from the catabolism of ketogenic amino acids and beta-oxidationof fatty acids. Three Acetyl-CoAs are used to produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is subsequently converted to mevalonate byHMG-CoA reductase. Mevalonate is used as a precursor in the formation ofisopentenyl pyrophosphate (IPP) and dimethylallyl-PP (DPP). In a head totail condensation, IPP and DPP are converted to geranyl-PP (GPP). GPPand an additional IPP undergo a condensation reaction to formfarnesyl-PP (FPP). In a head to head condensation reaction, two FPPmolecules are used to produce squalene. Squalene is cyclized by squalenesynthase to produce lanosterol, forming the tetracyclic steroid skeletonthat composes the cholestane ring. Following an additional nineteenenzymatic steps, lanosterol is converted to cholesterol. One key featureof cholesterol metabolism is that once squalene is cyclized and thecholestane ring is formed, the ring cannot be opened enzymatically inhuman cells. Consequently, it is not possible to reduce cholesterollevels by catabolism.

Lipid Transport

In most circumstances, the cellular demand for cholesterol is fulfilledby hepatic synthesis and cholesterol obtained from the diet. To deliverlipids from the site of intestinal absorption and hepatic synthesis,hydrophobic lipids (cholesterol, cholesterol esters, triglycerides (TG),and phospholipids) are packaged into lipoproteins for transport in theaqueous environment of the blood. In the small intestine, dietary lipidsare emulsified by bile, which aid the absorption of fats by intestinalepithelial cells (enterocytes) (FIG. 3). Within the enterocyte,cholesterol is converted into cholesterol esters and packaged into thecore of chylomicrons. The chylomicrons are released from the enterocytesby exocytosis, and transported through the lymphatic system which drainsinto the left subclavian vein. As chylomicrons travel through the blood,they deliver fatty acids (released from TGs by lipoprotein lipase) toadipose tissue and muscle. Ultimately, the chylomicrons remnants reachthe liver, where they are taken up by receptor-mediated endocytosis.When energy is in excess, the liver repackages dietary and newlysynthesized TG into very low-density lipoproteins (VLDLs). VLDLs, likechylomicrons, transport TG to adipose tissue and muscle. As aconsequence of TG removal by lipoprotein lipase, the VLDLs becomeintermediate density lipoproteins (IDLs). IDLs can either be taken up bythe liver where the constituents can be recycled into VLDLs, or IDLs canremain in the circulation where hepatic lipase and lipoprotein lipaseremove additional TGs. As VLDLs lose TGs the lipoprotein becomesenriched with cholesterol esters, and with further TG removal form lowdensity lipoproteins (LDLs). LDLs contain ApoB100 and are primarilycleared by the liver through LDL-receptor mediated endocytosis. Thecholesterol from LDLs can be used for either bile acid synthesis orrepackaged into nascent VLDLS for transporting additional TGs to adiposetissue and muscle. However, elevated levels of LDL contribute to thedevelopment of cardiovascular disease (CVD). Thus, current treatmentoptions for CVD work to lower serum LDL by targeting metabolic pathwaysresulting in increased expression of LDL-receptors that clear LDLs (i.e.statins).

Familial Hypercholesterolemia

Autosomal homozygous familial hypercholesterolemia (FH) is a raregenetic disease, which leads to the rapid onset of coronary heartdisease due to a persistent elevation in low density lipoprotein (LDL)cholesterol concentration (Fellin et al., 2015). Heterozygous FH is morecommon (1 in 200 to 1 in 500 people), effecting between 14 and 34million individuals worldwide (Nordestgaard et al., 2013). Patients withheterozygous FH demonstrate a clinical phenotype characterized byseverely elevated plasma levels of total cholesterol, low densitylipoprotein cholesterol, tendinous xanthomata, and have a highpredisposition for cardiovascular disease (Austin et al., 2004). Mostforms of familial hypercholesterolemia are genetic disorders disruptingnormal lipid metabolism, often due to mutations in genes encoding theLDL receptor (LDLR), apolipoprotein B-100 (apoB), or the proproteinconvertase subtilisin/kexin type 9 (PCSK9) (Robinson, 2013). Defects inany of these genes encoding proteins integral to lipoprotein metabolismresult in a significant increase in levels of low density lipoproteincholesterol (LDL-C) (Robinson, 2013). Homozygous FH is frequentlyassociated with the loss of LDL receptor expression or function.Although FH patients lack functional LDL receptors, uptake inmacrophages still occurs via scavenger receptors (SR), and lack of LDL-Rin other tissues yields more LDLs for macrophages. Therefore, FHpatients are at a greater risk for a myocardial infarction or stroke,which often occur within the first two decades of life (Fellin et al.,2015). Furthermore, these children do not respond to life stylemodification or statin therapy. Common treatment for homozygous FHcurrently depends on routine sessions of lipid apheresis (Lui et al.,2014). Homozygous FH is rare (˜1:1,000,000) and usually leads toadvanced CVD or death before the age of 20.

At the fundamental biochemical level, the increased risk ofcardiovascular disease is due to the inability of macrophages to clearcholesterol (Russell, 1992; Russell 2003, Russell, 2009). Interestingly,several enzymes endogenous to human cells are present that can likelydegrade the intermediate metabolites produced after ring opening. Forinstance, macrophages are equipped with a wide range of steroidogenicenzymes that can likely act on cholesterol once the ring has been opened(cholesterol hydrolases, CoA ligases, methyacyl-CoA racemases,branched-chain oxidases and acyltransferases, and a large family ofcytochrome p450s) (Enayetallah et al., 2008; Newman et al., 2005;Schiffer, 2015). This suggests that ring opening is the critical missingstep in preventing cholesterol accumulation. If macrophages wereequipped with the ability to rid the intima of LDL-C, the inflammatoryresponse would be reduced or even eliminated, and the formation ofatherosclerotic plaques may not occur. Our goal is to equip macrophageswith the metabolic capability to catabolize cholesterol, the criticalmissing step needed for the reduction and prevention of plaque formation(FIG. 4).

Bacterial Enzymatic Cholesterol Catabolism

The rationale for the proposed study is based on previous findingsrevealing the molecular mechanisms that allow Mycobacterium tuberculosissurvival within the phagosomes of macrophages during the choronic stageof infection (Pandey & Sassetti, 2008; Martens et al., 2008; Van derGeize et al., 2007). Tuberculosis (TB) is a chronic bacterial infection,typically affecting the lungs, caused by M. tuberculosis. Throughout thechronic stages of infection, M. tuberculosis avoids elimination by thehost immune response by residing intracellulary in alveolar macrophages(Ferrari et al., 1999; Russell, 2001; Stewart et al., 2003). As thebacterial infection progresses, the host immune system encases theinfected macrophages into dense granulomas structures. In many cases,the phagosomes of activated macrophages restrict the growth ofintracellular pathogens by preventing access to essential nutrients.However, M. tuberculosis has evolved mechanisms to obtain carbon fromhost cholesterol in effort to survive within the confined environment(Pandey & Sassetti 2008). This is achieved by M. tuberculosis throughthe activation of several operons, some of which encode genes thatenable cholesterol catabolism (Pandey & Sassetti, 2008; Van der Geize etal., 2007) (FIG. 5). The carbon obtained from host cholesterol is usedin lipid synthesis and the production of energy which aid the survivalof M. tuberculosis.

To date, our lab has identified key enzymes endogenous to Mycobacteriumtuberculosis (Brzostek et al., 2013), Sterolibacterium denitrificans(Chiang et al., 2008), Rhodococcus erythropolis (Petrusma et al., 2011)and Rhodococcus rhodocchrous (Petrusma et al., 2014) that catalyzecholestane ring opening. At the time the bacterial enzymes wereidentified, the field was still in the early stages of development andthe cholesterol catabolism pathway was not fully understood. There wascontroversy on whether the enzymes could catalyze cholestane ringopening with the side chain present (FIG. 6) (Penfield et al., 2014;Capyket et al., 2011; Chiang et al., 2008) or whether the enzymesrequired removal of the side chain prior to ring opening (FIG. 7)(Ouellet et al., 2011; Yeh et al., 2014; Petrusma et al., 2014). Toinitiate cholesterol degradation in human cells, it was critical for usto assess the minimal number of enzymes required to open the cholestaneB-ring and how the predicted catabolites (with and without thecholesterol side chain) effected enzyme activity. We humanized fourbacterial enzymes for this purpose.

The first enzyme required for ring opening is cholesterol dehydrogenase(CholD), a NAD(P)⁺ dependent dehydrogenase. CholD oxidizes the3β-hydroxyl at C3 of cholesterol (3β-hydroxycholest-5-ene) to yieldcholestenone (cholest-4-ene-3-one). Oxidation of the 3β-hydroxyl,producing a ketone at C3, also results in the isomerization of thedouble bond between C5 and C6 of ring B to C4 and C5 of ring A. The nexttwo enzymes in the cholesterol catabolism pathway the 3-keto groupformed by CholD. CholD was known to have activity with steroidsubstrates containing the cholesterol side chain suggesting CholD wasthe initiating step in cholesterol catabolism (Klink et al., 2013).

For the second step in catabolism, we identified two FAD+ dependent 3ketosteroid dehydrogenases (KstDs). One is 3-ketosteroidΔ¹-dehydrogenase (Δ¹-KstD) from R. erythropolis. Another is anoxiccholesterol catabolism B enzyme (acmB) from Sterolibacteriumdenitrificans. Both enzymes catalyze the desaturation of ring A byintroducing a double bond between the C1 and C2 atoms of 3-ketosteroidsubstrates. Δ¹-KstD is known to act on androstenedione(4-androstene-3,17-dione) a steroid molecule lacking the bulky sidechain. However, the Δ¹-KstD substrate specificity regarding thecholesterol side chain was not well established (Petrusma et al., 2011).Some reports indicated that Δ¹-KstD could accommodate substratescontaining the cholesterol side chain, while others suggested Δ¹-KstDactivity required prior side chain hydrolysis. Another report indicatedthe active site of the anoxic cholesterol metabolism B enzyme (acmB)from S. denitrificans could accommodate the cholesterol side chain(Chiang et al., 2008). If the active site of Δ¹-KstD did not utilizesteroid substrates with side chains, additional side chain cleavageenzymes would be necessary. Selecting an enzyme with the capacity to actupon steroid substrates with long side chains was desired to eliminatethe need for additional side chain cleavage enzymes.

The last enzyme required for ring cleavage, 3-ketosteroid 9α-hydroxylase(KshAB), is an NADH dependent Rieske-type oxygenase. KshAB is atwo-component iron-sulfur monooxygenase, consisting of a ferredoxinreductase (KshB) and a terminal oxygenase (KshA). KshAB catalyzes thehydroxylation of C9 on ring B of the cholesterol molecule. Previousreports suggest KshAB has subtle substrate specificity for3-ketosteroids, and can accommodate short side chains (Petrusma et al.,2009). Additionally, KshAB is able to act either before or after the C1and C2 dehydrogenation by 3-ketosteroid dehydrogenases.

The presence of: 1) the 3-keto group, 2) the isomerization of the doublebond between C4 and C5, 3) the desaturation of the C1 and C2 bond ofring A, and 4) the hydroxylation at C9 of ring B results in thedestabilization and spontaneous opening of the cholestane ring (FIG. 7).

Cholesterol Side Chain Removal and 3-Ketone Production

In the event the cholesterol side chain needed to be removed, wedeveloped a P450-FdxR-Fdx-P2A-3β-hydroxysteroid dehydrogenase (HSD2)bicistronic expression vector to produce two enzymes needed to removethe hydrophobic alkyl side chain and oxidize the 3β-hydroxy of sterolsubstrates. Using HSD2 to replace CholD also prevented the accumulationof cholestenone, which is toxic in high concentrations.

To produce these enzymes in human cells, we designed a fusion proteinconsisting of minimal structures from a human cytochrome P450 (CYP11Δ¹)and two electron transfer proteins, ferredoxin reductase and ferredoxin.This P450 fusion protein removes the side chain of cholesterol, yieldingpregnenolone. When the side chain is removed, human 3β-hydroxysteroiddehydrogenase (HSD2) can oxidize the 3β-hydroxyl of pregnenolone,yielding the 3-ketone product, progesterone. Along with the oxidation ofthe 3β-hydroxyl, HSD2 activity results in the isomerization of thedouble bond between C5 and C6 of ring B to C4 and C5 of ring A. HSD2cannot act on substrates that retain the C-17 side chain of cholesterol.Thus, the rate-limiting step for initiating cholestane ring opening viathis pathway is placed on the P450-FdxR-Fdx fusion protein. Overall wefelt this was the ideal scenario, as this placed the bottleneck fordegrading cholesterol on the P450 side chain cleavage enzyme, which alsoacts as the natural rate limiting enzyme in steroidogenesis.

In summary, we have shown that the bacterial enzymes necessary toinitiate cholesterol ring opening can be humanized and functionallyexpressed in human cells. First, we verified the enzymes were functionalby individually expressing each enzyme in E. coli and characterizing theactivity of the clarified bacterial lysate. Using RP-HPLC analysis weshowed that incubation of steroid substrates with each enzyme resultedin the formation of novel products with unique retention times andcharacteristic spectral properties that were not produced in the controllysates. Upon combining the bacterial lysates, we observed the formationof a new product with a unique retention time and a spectral shift thatwas indicative that ring opening had been achieved. The compoundsproduced by the clarified lysates were confirmed to be products ofsubstrate conversion by the use of C4-¹⁴C radiolabeled substrates.Because these compounds are not common, analytical standards of thepredicted intermediates are not readily available for comparison.Therefore, the compounds produced by the bacterial lysates were used asreferences for characterizing the activity of the transgenic human celllines. Each enzyme was independently expressed in Hep3B and U-937 cellsand the compounds that were produced matched the retention time andspectral properties of the products that were observed in the bacteriallysates. Additionally, the use of C4-¹⁴C radiolabeled substratesrevealed that both Hep3B and U-937 cells have endogenous metabolicactivity against cholesterol once the ring has been opened.

Applicants have identified four enzymes sufficient to initiatecholesterol ring opening in eukaryotic cells. Specifically, Applicantsdescribe designed and assembly of a pentacistronic expression vector(the cholesterol catabolizing cassette or CCC) that encodes for theenzymes needed to open the B-ring of cholesterol from a single openreading frame. We have generated U-937 cell lines that have beentransduced with lentivirus encoding the CCC. However, the size of theCCC insert may be at the upper limits for some types of lentiviralpackaging. Additionally, we are limited to a CMV promoter that may notbe ideal for expressing the CCC. In addition to lentiviral integration,the disclosed systems, methods, and compositions may use transposonmediated integration of the the disclosed cassettes, for example theCCC. Constructs may also have a responsive promoters (for example atet-responsive promoter) to provide for modulating expression of thedisclosed genes, such as the cholesterol catabolizing enzymes.

Although the prokaryotic and eukaryotic data are consistent, we need todefinitively show that the predicted intermediates are being produced.We are currently working towards demonstrating the mass spectrometryfragmentation patterns of the novel products produced by the bacteriallysate and human cells are identical. Once we have developed a referencelibrary for the predicted intermediates, our final goal will be toverify that U-937 macrophages expressing the CCC have the ability togenerate cholesterol ring opening from C2,3,4-¹³C3 cholesterol labeledLDLs.

LIST OF ABBREVIATIONS

Δ¹-KstD—3-ketosteroid Δ¹-dehydrogenase

3β-HSD—3β-hydroxysteroid dehydrogenase

3-HSC—3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one

3-HSP—3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione

9-OHCN—9-hydroxycholeste-4-ene-3-one (9-hydroxycholestenone)

9-OHCDN—9-hydroxycholeste-1,4-diene-3-one (9-hydroxycholestedieneone)

9-OHPD—9-hydroxy-pregn-4-ene-3,20-dione (9-hydroxyprogesterone)

9-OHPDD—9-hydroxy-pregn-1,4-diene-3,20-dione(9-hydroxyprogestedienedione)

AcmB—Anoxic cholesterol metabolism B enzyme

AUC—Area under curve

Bp—Base pair

CCC—Cholesterol catabolism cassette

CholD—Cholesterol dehydrogenase

CL—33-hydroxycholest-5-ene (cholesterol)

CDN—Choleste-1,4-diene-3-one (cholestedieneone)

CMV—Cytomegalovirus

CN—Choleste-4-ene-3-one (cholestenone)

CUC—Counts under curve

HP-THX—His patch thioredoxin

hr—hour

IMAC—Immobilized metal affinity chromatography

IPTG—Isopropyl-beta-D-thiogalactopyranoside

ISC—Iron-sulfur cluster

kD (kDa)—Kilodalton

KshAB—3-ketosteroid 9α-hydroxylase

LC-MS—Liquid Chromatography-Mass Spectrometry

mAU—Milli absorbance units

mg—Milligram

min—minute

mL—Milliliter

mM—Millimolar

nM—Nanomolar

nm—Nanometer

nt—Nucleotide

NTB—Nitrotetrazolium blue

OD600—Optical density at 600 nm

P2A—Porcine teschovirus 2A ribosomal skipping peptide

P450-FdxR-Fdx—P450 side chain cleavage enzyme-ferredoxinreductase-ferredoxin fusion protein

PAGE—Polyacrylamide gel electrophoresis

PD—Pregn-4-ene-3,20-dione (progesterone)

PDD—Pregn-1,4-diene-3,20-dione (progestedienedione)

PL—3β-hydroxypregn-5-en-20-one (pregnenolone)

PMS—Phenazine methylsulfate

PMSF—Phenylmethyulfonyl fluoride

PVDF—Polyvinylidene difluoride

RP-HPLC—Reverse phase high pressure liquid chromatography

SDS—Sodium dodecyl sulphate

T2A—Thosea asigna 2A ribosomal skipping peptide

TEV—Tobacco Etch Virus

μg—Microgram

μg—Microliter

μM—Micromolar

Cardiovascular disease (CVD), the leading cause of death, is responsiblefor one out of every three mortalities in the United States (Go et al.,2014). CVD is complex and often associated with aberations in normallipid metabolism, identified by elevated levels of low densitylipoproteins (LDLs) and/or reduced levels of high-density lipoproteins(HDLs) (Weverling-Rijnsburger et al., 2003). Atheroscleroticcardiovascular disease is characterized by arterial wall thickening andreduced arterial elasticity, resulting primarily from the chronicaccumulation of macrophages, engorged with cholesterol from lipoproteins(i.e. LDLs), within the intima of arteries (Singh et al., 2002) (FIG.1). For the majority of people, CVD is a progressive disease largelydependent on age and life style (Liu & Li, 2015); and can be managed bylowering low density lipoprotein cholesterol (LDL-C) with currentlyavailable treatment options (Franklin et al., 2014). In contrast,children affected by homozygous familial hypercholesterolemia (FH) areunresponsive to identical regimens. Familial hypercholesterolemia is agenetic disorder in which mutations in genes encoding LDL-receptorsprevent the expression of functional LDL receptors. LDL-receptors areintegral to lipoprotein metabolism and their loss result in a markedincrease in levels of LDL-C(Robinson, 2013). Elevations in serum LDL-Cplace FH patients at great risk for both myocardial infarction andstroke, which often occur within the first two decades of life (Fellinet al., 2015). To date, effective treatment options for patientssuffering from homozygous familial hypercholesterolemia have not beendeveloped.

Analysis has revealed that humans lack enzymes required to degrade thecholestane ring of cholesterol (Pandey & Sassetti, 2008; Martens et al.,2008; Van der Geize et al., 2007). Because humans lack enzymes needed toinitiate degradation, cholesterol accumulates when cellular uptakeexceeds efflux to passing high density lipoproteins. At a biochemicallevel this represents a critical component in the initiation of themaladaptive immune response that is responsible for the induction andprogression of atherosclerotic cardiovascular disease.

Recent studies have shown that M. tuberculosis is equipped with themetabolic capability to degrade cholesterol, which is used as a primaryenergy source in the phagosomes of macrophages (Russell, 1992; Russell2003, Russell, 2009). This observation raised the fundamental questionon whether macrophages could be engineered to degrade cholesterol byexpressing humanized bacterial cholesterol catabolizing enzymes.

In theory enabling cholesterol degradation in human macrophages wouldtransform the treatment of FH patients. The large number of variationsin genetic defects which culminate into the FH phenotype makes treatmentdifficult. Engineering human cells with the ability to degradecholesterol using methods reliant on recombinant gene expressionrepresents a novel approach in the management of both familialhypercholesterolemia and atherosclerotic cardiovascular disease.Developing such a therapy would circumvent the need for personalizedpharmacotherapies, and may serve as an entirely new treatment for allpatients suffering from atherosclerosis.

EXAMPLES

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description is to be regarded as illustrativein nature and not restrictive.

All references disclosed herein, whether patent or non-patent, arehereby incorporated by reference as if each was included at itscitation, in its entirety. In case of conflict between reference andspecification, the present specification, including definitions, willcontrol.

Although the present disclosure has been described with a certain degreeof particularity, it is understood the disclosure has been made by wayof example, and changes in detail or structure may be made withoutdeparting from the spirit of the disclosure as defined in the appendedclaims.

RESULTS AND DISCUSSION

Humanization and Cloning of CholD, acmB, Δ¹-KstD, and KshAB (Pro)

To characterize the cholesterol catabolizing enzymes, the genes encodingcholesterol dehydrogenase (CholD), anoxic cholesterol metabolism Benzyme (acmB), 3-ketosteroid-Δ¹-dehydrogenase (Δ¹-KstD), and3-ketosteroid 9α-hydroxylase (KshAB) were humanized. Humanization wasachieved by reverse translation of the bacterial amino acid sequencewith GeneOptimization (GeneArt) software, which predicts optimal codonusage, GC content, and adds a Kozak consensus sequence for H. sapiens.To aid cloning, “Gateway attachment sites” and restriction enzymerecognition sites were added, with the aid of GeneOptimization software.The cDNA of each humanized enzyme was then synthesized (GeneArt;Waltham, Mass. USA) and subcloned into standard commercial vectors asdiscussed in methods. To determine if the humanized enzymes were active,the enzymes were subcloned into Gateway expression vectors usingstandard Gateway cloning techniques. Humanized CholD, acmB, and Δ¹-KstDwere subcloned into pBADDest49 and heterologously expressed in Rosetta2E. coli, which contain plasmids that express tRNA that recognize codonusage that are common in humans but rare in E. coli. Two constructs forKshAB were designed to express this multi subunit enzyme in eitherprokaryotic or eukaryotic cells. The eukaryotic KshAB construct utilizesa Porcine teschnovirus-1 2A skipping peptide that allows for equimolarexpression of each subunit. Due to the bacterial ribosome beingunaffected by the viral 2A skipping peptide, a prokaryotic KshAB vectorwas designed as a bicistronic system that utilized two ShineDalgarnosequences positioned 5′ of both the A and B subunits. The prokaryoticKshAB construct was subcloned into pDest14 and heterologously expressedin C41 E. coli. Additionally, Rosetta2 and C41 E. coli were transformedwith the pUC19 vector to act as empty vector transformed controls.

CholD, acmB, Δ¹-KstD, and KshAB (pro) are Functional in E. coli

To characterize the products of steroid catabolism and verify thehumanized enzymes were indeed functional, reverse phase high pressureliquid chromatography (RP-HPLC) (FIGS. 8 and 9) methods were developed.As cholesterol is degraded, the resulting intermediates have increasedpolarity in comparison to the starting substrates. To effectivelyseparate and identify the downstream catabolites, we developed RP-HPLCmethods to increase the retention time of progesterone (t_(r)=13.8 min)and cholesterol (t_(r)=38.9 min) to an analytical C18 (octadecyl carbonchain bonded silica) column for sufficient lengths of time. This allowedus to separate the steroid bioconversion analytes, characterize theintermediates of cholesterol catabolism (identify retention times andspectral absorbance), and verify the enzymes were active.

Additionally, we needed to determine the substrate specificity of thecholesterol catabolizing enzymes in regards to the cholesterol sidechain. At the time there were conflicting reports in the literature. Thestudies of Penfield et al., Capyk et al., and Chiang et al., havesuggested that the enzymes within the cholesterol catabolism pathwayhave the ability to catabolize substrates with the hydrophobic sidechain (Chiang et al., 2008; Capyk et al., 2011; Penfield et al., 2014).Other reports indicated the ring opening enzymes required substrateswithout the hydrophobic side chain, suggesting side chain hydrolysisoccurred first (Ouellet et al., 2011; Petrusma et al., 2014; Yeh et al.,2014). To resolve this apparent conflict, we tested the substratespecificity of our humanized enzymes, using substrates with (cholesteroland cholestenone) and without (pregnenolone and progesterone) the C-17side chain.

Following transformation, bacterial cultures were grown, mechanicallylysed, and the crude protein was clarified by centrifugation. Theenzymatic activity of the clarified lysates was assessed by incubationwith the steroid substrates cholesterol (CL, 30 hydroxycholest-5-ene),cholestenone (CN, choleste-4-ene-3-one), pregnenolone (PL, 30hydroxypregn-5-en-20-one) or progesterone (PD, pregn-4-ene-3,20-dione).Reactions were stopped by extracting with ethyl acetate and the steroidbioconversion analytes were analyzed by RP-HPLC. In agreement with theliterature, E. coli were found to be ideal organisms for steroidbioconversion analysis due to their lack of metabolic activity againststeroid substrates, as observed with pUC19 transformed E. coli incubatedwith 100 μM cholesterol (FIG. 10), cholestenone (FIG. 11), pregnenolone(FIG. 12), or progesterone (FIG. 13) for 24 hours. The use of E. colifacilitated the identification of novel metabolites generated by thehumanized cholesterol catabolizing enzymes through observation of newabsorbances/peaks with unique retention times following incubation ofthe substrates with the enzyme expressing bacterial lysates.Additionally, novel metabolites were confirmed as products of enzymaticsteroid bioconversions by C4-¹⁴C scintillation events when cholesterol(CL) or progesterone (PD) were used.

The CholD enzyme was determined to be active by incubating the bacteriallysate with two 30-hydroxy steroid substrates in independent reactionscontaining 100 μM C4-¹⁴C labeled cholesterol (CL) (FIG. 14) or 100 μMpregnenolone (PL) (FIG. 15) for 24 hours. Both cholesterol andpregnenolone lack an observable UV absorbance within the 200-300 nmrange, a feature that results from a lack of conjugation in the A-ringof the cholestane ring. However, following oxidation of the 3β-hydroxyto a 3-ketone with concomitant isomerization of the C5-C6 double bond toC4-C5, the resulting conjugated 3-ketosteroids (cholestenone andprogesterone) possess a detectable UV absorbance. Although a lack in UVabsorbance prevents the determination of the substrates retention time,we used radiolabeled cholesterol and an in-line flow scintillationanalyzer to determine the retention time of the substrate. Analysis ofthe CholD bacterial lysate incubated with 100 μM C4-¹⁴C labeledcholesterol (CL) showed reduction in CL (λ_(max): <200 nm; t_(r)=38.9min) and formation of a new peak corresponding to cholestenone (CN)(λ_(max): 239 nm; t_(r)=36.9 min) within 24 hours (FIG. 14 panels b &c). Cholestenone (CN) demonstrated a unique retention time of 36.9minutes and a lambda max of 239 nm that was not observed in the controlpUC19 bacterial lysate incubated with 100 μM cholesterol (CL) for 24hours. Analysis of the cholestenone (CN) UV absorbance spectrum showsthe λ_(max) of the 36.9 minute peak is 239 nm (FIG. 14 panel d),matching the UV absorbance and retention time of the cholestenone (CN)analytical standard. Analysis of C4-¹⁴C scintillation events confirmedthat production of radiolabeled cholestenone (CN) is concomitant to thereduction of C4-¹⁴C cholesterol (CL) (FIG. 14 panel c). In addition,incubating the CholD bacterial lysate with 100 μM pregnenolone (PL)(λ_(max): <200 nm; t_(r)=15.5 min) resulted in the formation ofprogesterone (PD) (λ_(max): 245 nm; t_(r)=13.8 min) within 24 hours(FIG. 15). Progesterone (PD) demonstrated a unique retention time of13.8 minutes and a lambda max of 245 nm that was not observed in thecontrol pUC19 bacterial lysate incubated with 100 μM pregnenolone (PL)for 24 hours (FIG. 15 panels b). Analysis of the progesterone (PD) UVabsorbance spectrum shows the λ_(max) of the 13.8 minute peak is 245 nm(FIG. 15 panels c & d), matching the UV absorbance and retention time ofthe progesterone (PD) analytical standard. This data confirms thathumanized CholD can be heterologously expressed in E. coli as an activeenzyme, and its activity is not hindered by the presence or the absenceof the cholesterol side chain.

The acmB enzyme was determined to be active by incubating the bacteriallysate with two 3-ketosteroid substrates in independent reactionscontaining 100 μM cholestenone (CN) (FIG. 16) and 100 μM progesterone(PD) (FIG. 17) for 24 hours. Both the substrates and their respectiveproducts demonstrate an observable UV absorbance, facilitating thedetermination of their retention times. AcmB is a dehydrogenase thatcatalyzes the ring-A C1-C2 desaturation of 3-ketosteroid substrates.Analysis of the acmB bacterial lysate following incubation with 100 μMcholestenone (CN) (λ_(max): 239 nm; t_(r)=36.9 min) for 24 hours revealsthe formation of choleste-1,4-diene-3-one (CDN) (λ_(max): 241 nm;t_(r)=29.8 min) (FIG. 16). Choleste-1,4-diene-3-one (CDN) demonstrated aunique retention time of 29.8 minutes (FIG. 16 panel b) and a lambda maxof 241 nm (FIG. 16 panel c) that was not observed in the control pUC19bacterial lysate incubated with 100 μM cholestenone (CN) for 24 hours.In addition, incubating the acmB bacterial lysate with 100 μM C4-¹⁴Clabeled progesterone (PD) (λ_(max): 245 nm; t_(r)=13.8 min) resulted inthe formation of C4-¹⁴C labeled pregn-1,4-diene-3,20-dione (PDD)(λ_(max): 247 nm; t_(r)=10.0 min) (FIG. 17). Pregn-1,4-diene-3,20-dione(PDD) demonstrated a unique retention time of 10.0 minutes (FIG. 17panel b) and a lambda max of 247 nm (FIG. 17 panel d) that was notobserved in the control pUC19 bacterial lysate incubated with 100 μMC4-¹⁴C labeled progesterone (PD) for 24 hours. Analysis of C4-¹⁴Cscintillation events confirmed that production of radiolabeledpregn-1,4-diene-3,20-dione (PDD) is concomitant to the reduction ofC4-¹⁴C progesterone (PD) (FIG. 17 panel c). This data confirms thathumanized acmB can be heterologously expressed in E. coli as an activeenzyme, and its activity is not hindered by the presence or absence ofthe cholesterol side chain.

The Δ¹-KstD enzyme, a second 3-ketosteroid dehydrogenase that catalyzesthe same reaction as acmB, was determined to be active by incubating thebacterial lysate with the 3-ketosteroid substrates, 100 μM cholestenone(CN) (FIG. 18) and 100 μM C4 ¹⁴C labeled progesterone (PD) (FIG. 19) for24 hours. Analysis of the Δ¹-KstD bacterial lysate following incubationwith 100 μM cholestenone (CN) (λ_(max): 239 nm; t_(r)=36.9 min) for 24hours reveals an inability to produce choleste-1,4-diene-3-one (CDN)(λ_(max): 241 nm; t_(r)=29.8 min) (FIG. 18) as observed with the acmBbacterial lysate. A lack in activity with cholestenone (CN) suggeststhat Δ¹-KstD may not accommodate the cholesterol side chain. However,incubating the Δ¹-KstD bacterial lysate with 100 μM C4-¹⁴C labeledprogesterone (PD) (λ_(max): 245 nm; t_(r)=13.8 min) resulted in theformation of C4-¹⁴C labeled pregn-1,4-diene-3,20-dione (PDD) (λ_(max):247 nm; t_(r)=10.0 min) (FIG. 19). The pregn-1,4-diene-3,20-dione (PDD)product demonstrated the same retention time of 10 minutes (FIG. 19panel b) and lambda max of 247 nm (FIG. 19 panel d) as seen with thepregn-1,4-diene-3,20-dione (PDD) formation with the acmB lysate.Analysis of C4-¹⁴C scintillation events confirmed that production ofradiolabeled pregn-1,4 diene-3,20-dione (PDD) is concomitant to thereduction of C4-¹⁴C progesterone (PD) (FIG. 19 panel c). This dataconfirms that humanized Δ¹-KstD can be heterologously expressed in E.coli as an active enzyme, but its activity is hindered by the presenceof the cholesterol side chain.

The KshAB enzyme was determined to be active by incubating the bacteriallysate with 100 μM cholestenone (CN) (FIG. 20) and 100 μM C4-¹⁴C labeledprogesterone (PD) (FIG. 21) for 24 hours. KshAB is a hydroxylase thatcatalyzes the addition of a hydroxyl group to the ring-B C9 of3-ketosteroids. Analysis of the KshAB bacterial lysate followingincubation with 100 μM cholestenone (CN) (λ_(max): 239 nm; t_(r)=36.9min) for 24 hours reveals a small formation of9-hydroxycholeste-4-ene-3-one (9 OHCDN) (λ_(max): 239 nm; t_(r)=8.9 min)(FIG. 20). The 9-hydroxycholeste-4-ene-3-one (9 OHCDN) productdemonstrated a unique retention time of 8.9 minutes (FIG. 20 panel b)and a lambda max of 239 nm (FIG. 20 panel c) that was not observed inthe control pUC19 bacterial lysate incubated with 100 μM cholestenone(CN) for 24 hours. Incubation of the KshAB bacterial lysate with 100 μMC4-¹⁴C labeled progesterone (PD) (λmax: 245 nm; t_(r)=13.8 min) resultedin the formation of C4-¹⁴C labeled 9-hydroxypregn-4-ene-3,20-dione(9-OHPD) (λ_(max): 245 nm; t_(r)=5.2 min) (FIG. 21). The9-hydroxypregn-4-ene-3,20-dione (9-OHPD) product demonstrated a uniqueretention time of 5.2 minutes (FIG. 21 panel b) and a lambda max of 245nm (FIG. 21 panel d) that was not observed in the control pUC19bacterial lysate incubated with 100 μM C4-14C labeled progesterone (PD)for 24 hours. Analysis of C4-¹⁴C scintillation events confirmed thatproduction of radiolabeled 9-hydroxypregn4-ene-3,20-dione (9-OHPD) isconcomitant to the reduction of C4-¹⁴C progesterone (PD) (FIG. 21 panelc). This data confirms that humanized KshAB can be heterologouslyexpressed in E. coli as an active enzyme, but its activity is moderatelyaffected by the presence of the cholesterol side chain.

To characterize the production of3-hydroxy-9,10-secopregn-1,3,5(10)-triene9,20-dione (3-HSP), thebacterial lysates expressing acmB (FIG. 22) or Δ¹-KstD (FIG. 23) werecombined with KshAB and incubated with 100 μM C4-14C labeledprogesterone (PD) (λ_(max): 245 nm; t_(r)=13.8 min) for 24 hours.Reactions containing acmB and KshAB (FIG. 22) or Δ¹-KstD and KshAB (FIG.23) resulted in exhaustion of the progesterone (PD) substrate withconcomitant formation of a new product with a unique retention time of7.2 minutes and a lambda max of 280 nm that was not observed in thecontrol pUC19 bacterial lysate following incubation with 100 μM C4-¹⁴Clabeled progesterone (PD) for 24 hours. Analysis of C4-¹⁴C scintillationevents confirms that production of radiolabeled 3-HSP is concomitant tothe reduction of C4-¹⁴C progesterone (PD) (FIG. 22 panel c and 23 panelc). This data confirms that the combined activities of humanized acmBand KshAB or Δ¹-KstD and KshAB lead to the formation of the novelmetabolite, 3-HSP.

Next, we wanted to determine whether the combined activities of CholD,acmB, Δ¹-KstD, and KshAB could facilitate ring opening starting from thesubstrate cholesterol. In two separate reactions, CholD, acmB, and KshAB(FIG. 24) or CholD, Δ¹-KstD, and KshAB (FIG. 25) were combined andincubated with 100 μM C4-¹⁴C labeled cholesterol (CL) (λ_(max): <200 nm;t_(r)=38.9 min) for 24 hours. Analysis of the combined CholD, acmB, andKshAB lysates (FIG. 24) shows formation of cholestenone (CN) (λ_(max):239 nm; t_(r)=36.0 min), choleste-1,4-diene-3-one (CDN) (λ_(max): 241nm; t_(r)=29.5 min), and3-hydroxy-9,10-secocholestene-1,3,5(10)-triene-9-one (3-HSC) (λ_(max):280 nm; t_(r)=5.3 min) within 24 hours. Analysis of C4-¹⁴C scintillationevents confirms production of radiolabeled 3-HSC is concomitant to thereduction of C4-¹⁴C labeled cholesterol (CL) (FIG. 24 panel c). Incontrast, analysis of the CholD, Δ¹-KstD, and KshAB lysates (FIG. 25)shows formation of cholestenone (CN) (λ_(max): 239 nm; t_(r)=36.0 min)and 9-hydroxycholeste-4-ene-3-one (9-OHCN) (λ_(max): 239 nm; t_(r)=8.9min), but not choleste1,4-diene-3-one (CDN) (λ_(max): 241 nm; t_(r)=29.5min) or 3-hydroxy-9,10-secocholestene1,3,5(10)-triene-9-one (3-HSC)(λ_(max): 280 nm; t_(r)=5.3 min) within 24 hours. Additionally, analysisof C4-¹⁴C scintillation events confirms Δ¹-KstD lacks the ability todesaturate cholestenone (CN), as previously demonstrated (side chainissues), and thus 3 HSC cannot be generated (FIG. 25 panel c). From thisdata, we have determined that CholD, acmB, and KshAB equip the bacteriallysates with the ability to oxidize the 30-hydroxyl to a 3-ketone,desaturate the C1-C2 bond of ring-A, and hydroxylate the ring-B C9 ofCL, respectively. The presence of all three humanized enzymes equip thebacterial lysates with the ability to produce 3-HSC, a novel compoundhaving a unique λ_(max) and t_(r) that is not observed in the controlpUC19 lysate incubated with cholesterol (CL) for 24 hours. If Δ¹-KstD isto be used to generate ring opening, removal of the cholesterol sidechain may be required.

Cholestenone is Toxic to Cells

A key step in developing the enzyme cassette was to determine whichenzymes were sufficient in activity to avoid creating bottlenecks thatled to the accumulation of toxic intermediates. Cholesterol ring openingmay require the 3β-hydroxyl of cholesterol to be oxidized to a 3-ketoneby CholD, forming the product cholestenone. Cholestenone, anintermediate degradation product in 3-HSC formation, is known topartition into the plasma membrane and disrupt normal function. Todetermine the level of toxicity associated with cholestenone, thecompound resazurin was used to construct a cell viability curve withHep3B cells in the presence of increasing concentrations of cholestenone(FIG. 26). The results demonstrate that cholestenone concentrationsabove 60 μM become detrimental to cell viability. Thus, it is importantthat cholestenone does not accumulate. To maintain low levels ofcholestenone, it is pertinent for the downstream enzymes to be highlyefficient in catalyzing their respective reactions. Unfortunately, wefound that the activities of acmB and KshAB expressed in eukaryoticcells were not sufficient to maintain low levels of cholestenone, likelydue to substrate accessibility issues (data not shown). The toxicityassociated with cholestenone is due to the presence of the ring-A3-ketone and the hydrophobic cholesterol side chain. The 3 ketone isrequired by the remaining bacterial enzymes to catalyze ring opening andis therefore a necessary catalytic step. However, as demonstrated in thebacterial lysates, the presence of the cholesterol side chain is notrequired to produce ring opening. Therefore, to reduce the toxicityassociated with generating intermediates of cholesterol degradation, wedecided to explore side chain removal options using the human P450FdxR-Fdx fusion protein.

P450-FdxR-Fdx-P2A-HSD2 Expression in U-937-Derived Macrophages

The P450-FdxR-Fdx-P2A-HSD2 construct encodes for two separate enzymes.The first enzyme, P450-FdxR-Fdx is a fusion protein consisting of theP450 cholesterol side chain cleavage enzyme (CYP11A), ferredoxinreductase, and ferredoxin. The P450-FdxR-Fdx was modeled after the P450F2 system designed by the Miller lab (Huang & Miller, 2001). Followingthe P450-FdxR-Fdx is a Porcine teschovirus-1 2A skipping peptide forco-expression of the second enzyme, 30-hydroxysteroid dehydrogenase 2(HSD2). The P450-FdxR-Fdx fusion protein catalyzes the conversion ofcholesterol into pregnenolone by removal of the cholesterol side chainthrough three monooxygenase reactions. Once the side chain has beenremoved, HSD2 can oxidize the 30-hydroxyl of pregnenolone to a 3-ketoneto form progesterone. This enzyme construct replaces the need for thebacterial enzyme CholD, and solves the issues of toxicity associatedwith cholestenone by removing the hydrophobic side chain. To verify theenzyme construct was functional, stable U-937 cell lines were generatedusing lentivirus as described in methods. U-937 cells are a humanleukemic monocyte lymphoma cell line that can be stimulated todifferentiate into macrophages. Macrophages express low densitylipoprotein receptors (LDL-R) and scavenger receptors (SR), andtherefore have the ability to take up extracellular cholesterol in aphysiological manner (FIG. 27). Additionally, macrophages are the targetcell line we plan to engineer with the cholesterol catabolizing cassetteto act as a cellular vehicle for the amelioration of atheroscleroticplaques.

The two enzymes in the P450-FdxR-Fdx-P2A-HSD2 construct were determinedto be active by incubating the transgenic U-937-derived macrophages with50 μg C4-¹⁴C cholesterol labeled LDLs (163 nCi C4-¹⁴C cholesterol) for72 hours (FIG. 28). RP-HPLC analysis reveals the P450-FdxR-Fdx-P2A-HSD2macrophages are equipped with the ability to hydrolyze the cholesterolside chain and oxidize the 30-hydroxyl to a 3-ketone formingprogesterone (PD) (t_(r)=13.8 min, λ_(max) 245 nm) following 72 hoursincubation (FIG. 28 panels b, d, & f). In contrast, control macrophageslacked the ability to convert cholesterol to progesterone (FIG. 28panels a, c, & e).

To determine whether the P450-FdxR-Fdx or HSD2 was the rate limitingenzyme in the conversion of cholesterol to progesterone, theP450-FdxR-Fdx-P2A-HSD2 macrophages were incubated with 15.8 μg (10 μM)pregnenolone (PL) (λ_(max): <200 nm; t_(r)=15.5 min) for 72 hours (FIG.29). Analysis revealed a robust conversion of pregnenolone (PL) toprogesterone (PD) (t_(r)=13.8 min, λ_(max) 245 nm) by theP450-FdxRFdx-P2A-HSD2 expressing macrophages following the 72 hoursincubation (FIG. 29 panels b & d). In contrast, control macrophageslacked the ability to convert pregnenolone (PL) to progesterone (PD)(FIG. 29 panels a & c).

These results suggest that the rate limiting enzyme in the conversion ofcholesterol to progesterone is the P450-FdxR-Fdx fusion protein. Allsubsequent catalytic steps may require side chain removal prior toopening the cholesterol ring. This may place a bottleneck for degradingcholesterol at the first enzymatic step, the removal of the cholesterolside chain by the P450-FdxR-Fdx fusion protein, and thus regulates theremaining enzymes ability to participate in generating cholesterol ringopening.

Purification of Δ¹-KstD

To better characterize one of the downstream enzymes in 3-HSPproduction, Δ¹-KstD was isolated and its kinetic parameters weredetermined (Outline of isolation to kinetic analysis of Δ¹-KstD FIG.30). As previously described, Δ¹-KstD was heterologously expressed inRosetta2 E. coli as an N-terminal His-Patch thioredoxin fusion protein.Following verification Δ¹-KstD could be functionally expressed in E.coli, the His-Patch thioredoxin fusion protein (HP-THX) was partiallypurified by immobilized metal affinity chromatography (IMAC) using animidazole linear gradient (FIG. 31). The chromatogram, monitoringprotein absorption at 280 nm, shows the majority of endogenous E. coliproteins eluting in the flow through fractions and a single elongatedpeak eluting in the linear gradient between 60 and 170 mM imidazole.Relative dehydrogenation activities of the major fractions collectedfrom IMAC were assessed using an in-gel nitrotetrazolium blue assay(NTB) (FIG. 32). The in-gel nitrotetrazolium blue activity assayrevealed high amounts of Δ¹-KstD activity in the lysate and elutionfractions 19, 20, 21, and 22 (FIG. 33).

A coomassie stained SDS-PAGE of the lysate, flow through, washes, andelution fractions was made to assess the purity of the collectedfractions from IMAC (FIG. 34). The lane containing the clarified lysateshows the total cellular protein. As seen in the chromatogram, amajority of the endogenous E. coli protein was unable to bind to theNi²⁺ chelating column and eluted in the flow through fractions (FTfractions 1-4). The wash fractions demonstrate that the non-boundprotein was removed prior to the protein with highest affinity eluted inthe linear gradient between 60-170 mM imidazole (elution fractions19-27). The coomassie stained SDS-PAGE shows the 100 kDa HP-THX-Δ¹-KstDfusion protein eluting over several fractions of the imidazole gradient.Δ¹-KstD was engineered with an N-terminal FLAG tag to identify theenzyme. The anti-FLAG western blot recognized the 100 kDa protein bandas the FLAG tagged HP-THX-Δ¹-KstD fusion protein. In addition, a numberof lower molecular weight proteins were identified, likely resultingfrom C-terminal degradation products of Δ¹-KstD (FIG. 35). Elutionfraction 21 was assessed to contain a high yield of Δ¹-KstD, relativelylow contaminating proteins, and had sufficient activity for furthercharacterization. The concentration of Δ¹-KstD captured in elutionfraction 21 was estimated to contain 0.385 mg/mL of Δ¹-KstD with 79.6%purity determined by densitometry from the Coomassie stained SDS-PAGE(FIG. 36). The HP-THX-Δ¹-KstD captured in elution fraction 21 was elutedin 25 mM Tris-HCl, pH 7.5, containing 500 mM NaCl and 120 mM imidazole.

To validate Δ¹-KstD was responsible for the activity observed in the NTBassay, 0.77 μg of the partially purified protein was incubated for fourhours at 37° C. with the substrate progesterone (PD). Followingincubation, the reaction was extracted and analyzed by RP-HPLC (FIG.37). The RP-HPLC analysis revealed a diminished absorbance maximum atthe retention time typical of the substrate progesterone (PD) (λ_(max):245 nm, t_(r)=13.8 min) and formation of the productpregn-1,4-diene-3,20-dione (PDD) (λ_(max): 247 nm, t_(r)=10.0 min).Within four hours, Δ¹-KstD converted approximately 90% of theprogesterone (PD) substrate to pregn-1,4-diene-3,20-dione (PDD). Thisdata demonstrates that the enzyme isolated from IMAC is in fact Δ¹-KstDand is highly active.

Measurement of Δ¹-KstD Activity Using Progesterone (PD) and Resazurin

To determine Δ¹-KstD's kinetic parameters, I developed a directlycoupled fluorometric assay using the compound resazurin. Resazurin, aweakly fluorescent redox dye, is irreversibly reduced upon acceptingprotons released from a donor molecule. Reduction of resazurin resultsin the formation of the highly fluorescent product resorufin. In thisassay, protons are removed from the 3-ketosteroid substrates ring-AC1-C2 bond by Δ¹-KstD. The Δ¹-KstD FADH cofactor donates the protons toresazurin resulting in the formation of resorufin (FIG. 38). Thisreduction can be measured by monitoring the increase in fluorescenceintensity with time, allowing the assessment of the initial rates ofΔ¹-KstD substrate conversion. To demonstrate the linearity of thisassay, a standard curve was made by adding several concentrations ofresorufin to inversely proportional concentrations of resazurin (FIG.39).

Several concentrations of Δ1-KstD (0.05, 0.19, 0.37, 0.55, 1.1, 1.6,2.12 nM) were assayed to determine the optimal concentration of enzymerequired to measure the linear phase of the reaction (FIG. 40). Theresults show a linear increase in fluorescence with all testedconcentrations over 3.5 minutes. Extending the analysis further than 3.5minutes resulted in a decrease in linearity as enzyme concentration wasincreased. Enzyme progress curves were made by incubating 1.6 nM (FIG.41), 1.1 nM (FIG. 42), and 0.55 nM (FIG. 43) Δ¹-KstD with increasingconcentrations of progesterone (1, 2.5, 5, 10, 20, 30, 40 μM). Theenzyme progress curves using 0.55 nM Δ¹-KstD demonstrated the highestlinearity and were used for further analysis. The slopes of each curvewere fit to a hyperbola using nonlinear regression (FIG. 44). Δ¹-KstD'sK_(m) (8.3+/−0.5 mM) and V_(max) (2.2+/−0.05 RFU/sec) were determined byfitting the data to the Michaelis-Menten equation. Steady-state kineticsdemonstrate Δ¹-KstD is sufficiently active in terms of physiologicalconditions with progesterone; the expected metabolic intermediate withinthe cholesterol catabolism pathway.

In addition to determining the kinetic parameters of Δ¹-KstD with thesubstrate progesterone, the resazurin assay was used to screen anadditional 20 cholesterol derivatives. The Δ¹-KstD substrate screenprovided insight on the enzymes substrate specificity in regards tovariations to the steroid nucleus and side chain. The cholesterolderivatives included a number of pregnane-, androstane-, andcholestane-based compounds. Data shows the percent activity of the 20screened substrates compared to progesterone (FIG. 45). Of the 21compounds tested, Δ¹-KstD demonstrated activity with nine substrates(ranked highest to lowest in activity) (FIG. 46): progesterone, 17hydroxyprogesterone, 11-deoxycorticosterone, testosterone, cortisone,androstenedione, spironolactone, dihydrotestosterone, and testosteroneenanthate. Substrates resulting in less than 10% activity compared toΔ¹-KstD's activity with progesterone were considered poor substrates(FIG. 47). Results demonstrate that Δ¹-KstD requires the 3-ketone onring-A; Δ¹-KstD specificity exceeds that of the previously establishedsubstrate, androstenedione (i.e. progesterone); however, Δ¹-KstD lacksthe capability to utilize substrates with long alkyl C17 side chains(i.e. cholestenone).

Δ¹-KstD Expression in Hep3B and U-937 Cells

The original Δ¹-KstD construct expressed poorly in eukaryotic cells dueto the Kozak consensus sequence being positioned between a 6× His tagand tetracysteine tag (Appendix A15). For optimal expression of Δ¹-KstDin eukaryotic cells, we modified the original construct by removing theTEV site, 6× His tag, and tetracysteine tag. Using Gibson assembly and arepair string encoding a new Kozak consensus sequence, the Δ¹-KstDconstruct was repaired (FIG. 48). The repaired Δ¹-KstD construct wassubcloned into two lentiviral expression vectors which were used togenerate several stable PGK and CMV driven Δ¹-KstD Hep3B cells lines.Expression levels of the PGK and CMV driven Δ¹-KstD were assessed bywestern blot (FIG. 49). A high level of expression was observed in the3×CMV Δ¹-KstD cell line, and was subsequently used to assess enzymeactivity. Δ¹-KstD expressing Hep3B cells and control non-transducedHep3B cells were grown to confluency in 60 mm dishes and incubated with15.7 μg (10 μM) progesterone (PD) spiked with 100 nCi C4-¹⁴Cradiolabeled PD (FIG. 50). Following 24, 48, and 72 hours incubation,the cells and media were extracted with ethyl acetate, and the lipidprofiles were analyzed by RP-HPLC. Spectral data from the RP-HPLCanalysis of Δ¹-KstD Hep3B cells revealed pregn-1,4-diene-3,20-dione(PDD; t_(r)=10.0 minutes; λ_(max) 247 nm) accumulated over the 72 hourtime course (FIG. 51 panel b). The retention time and lambda max of the10.0 minute peak matches the PDD peak identified in the Δ¹-KstDbacterial lysate and partially purified Δ¹-KstD incubated with PD. Asexpected, Hep3B control cells lacked the metabolic activity to producePDD, as observed by the absence of a 10.0 minute peak (FIG. 51 panel a).Quantitative analysis of the area under the curve shows that as PD isutilized by Δ¹-KstD Hep3B cells (FIG. 52b ), a concomitant formation ofPDD is observed (FIG. 52 panel d). The decrease in the PD substrate withHep3B control cells (FIG. 52 panel a) can be explained by analysis ofthe ¹⁴C scintillation events (FIG. 53 panel a).

Unsurprisingly, Hep3B cells have endogenous metabolic capability tometabolize PD (i.e. bile acid formation, etc). More importantly, theC4-¹⁴C PDD peak identified with the Δ¹-KstD Hep3B cells (FIG. 53 panelb) did not appear in the Hep3B control samples (FIG. 53 panel a).Quantitative analysis of the counts under the curve shows that as theradiolabeled PD substrate is utilized by Hep3B cells expressing Δ¹-KstD,the radiolabeled PDD product forms with time (FIG. 54).

We next set out to characterize the activity of our Δ¹-KstD expressingHep3B cells against the primed ring opening substrate,9-hydroxypregn-4-ene-3,20-dione (9-OHPD). The 9-OHPD substrate (λ_(max)245 nm; t_(r)=5.2 min) was enzymatically produced by incubation ofprogesterone (PD) with the KshAB bacterial lysate (FIG. 55). Followinghydroxylation of PD by KshAB, this 3-ketosteroid product requires thedesaturation of the C1-C2 bond of ring-A by Δ¹-KstD to form the unstableproduct, 9-hydroxypregn-1,4-diene-3,20-dione (9-OHPDD). The B-ring of9-OHPDD is then subject to spontaneous non-enzymatic cleavage withconcomitant aromatization of ring-A to form the product 3hydroxy-9,10-secopregn-1,3,5(10)-triene-9,17-dione (3-HSP). As aconsequence of ring-A aromatization, 3-HSP demonstrates a characteristiclambda max of 280 nm and is an indicator that ring opening has beenachieved. To study whether the Δ¹-KstD Hep3B cells could catalyze thisreaction if presented with the appropriate intermediate, cells wereincubated with 17 μg (10 μM) of 9-OHPD for 72 hours. RP-HPLC analysis ofthe Δ¹-KstD Hep3B cells at λ 245 nm shows 9-OHPD decrease over the timecourse, whereas Hep3B control cells retain the 9-OHPD substrate (FIG.56). RP-HPLC analysis at λ 280 nm shows that as 9-OHPD is catabolized bythe Δ¹-KstD Hep3B cells, a new peak with a retention time of 7.2 minutesand a lambda max of 280 nm appears with time (FIG. 57 panel b). Anidentical peak, corresponding to 3-HSP, was observed with pregnane ringopening by Δ¹-KstD and KshAB clarified bacterial lysates followingincubation with progesterone (PD). In contrast, non-transduced Hep3Bcells did not demonstrate the ability to produce the same 7.2 minutepeak when incubated with 9-OHPD (FIG. 57 panel a). Quantitative analysisof the 9-OHPD area under the curve shows that Hep3B control cells lackthe metabolic activity to metabolize 9-OHPD, as revealed by theretention of the substrate over the 72 hour time course. In comparison,Δ¹-KstD Hep3B cells catabolized the 9-OHPD substrate leading to theproduction of 3-HSP. Maximal production of 3 HSP was observed at 48hours. At later time points, 3-HSP peak area was found to decrease.Reduction in 3-HSP suggest that Hep3B cells have endogenous metaboliccapability to further modify the cholestane ring once opened (FIG. 58).

We have also verified that Δ¹-KstD can be independently expressed inU-937 cells (FIG. 59). As described in methods, stable Δ¹-KstDexpressing monocytes were generated. The Δ¹-KstD U-937-derivedmacrophages were incubated with 15.7 μg (10 μM) progesterone (PD) spikedwith 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min; λ_(max) 245 nm) for 72hours. Analysis of U-937 Δ¹-KstD cells shows formation of a new peakwith a retention time of 10.0 minutes, a λ_(max) of 247 nm, andcontaining C4-14C scintillation events corresponding topregn-1,4-diene-3,20-dione (PDD; t_(r)=10.0 min; λ_(max) 247 nm)following 72 hours incubation with PD (FIG. 59 panels b, d, & f). Incontrast, U-937 control cells lack the ability to catabolize PD to PDD,as seen by the absence of a peak with a 10.0 minute retention time (FIG.59 panels a, c, & e).

To confirm these cells could generate 3-HSP when provided theappropriate intermediate substrate, the Δ¹-KstD U-937-derivedmacrophages were incubated with 17 μg (10 μM)9-hydroxypregn-4-ene-3,20-dione (9-OHPD, t_(r)=5.2 min; λ_(max) 245 nm)produced and isolated from bacterial KshAB lysate. Analysis of Hep3BΔ¹-KstD cells following 72 hours incubation shows the formation of thering opened product, 3-HSP (t_(r)=7.2 min; λ_(max) 280 nm) (FIG. 60panels b & d). In contrast, control Hep3B cells lack the metaboliccapability to produce 3-HSP (FIG. 60 panels a & c).

KshAB Expression in Hep3B and U-937 Cells

As previously described in methods, two KshAB vectors were designed forprokaryotic and eukaryotic expression. The prokaryotic KshAB expressionvector was designed using conventional methods to coexpress the A and Bsubunits using two Shine-Dalgarno sequences. To coexpress KshAB ineukaryotic cells, we designed a bicistronic expression vector byinserting DNA encoding the Porcine teschovirus-1 2A skipping peptidebetween the A and the B subunit genes. The 2A skipping peptide consistsof 22 amino acids that facilitate equimolar expression of two or moregenes from one expression vector. The C-terminus of the 2A skippingpeptide contains two proline residues separated by a glycine. This motifadopts a confirmation in the ribosomal exit tunnel that interferes withthe ability of the ribosome to synthesize the nascent polypeptidestring. In efforts to continue polypeptide synthesis, the ribosome skipsformation of the peptide bond leaving twenty-one amino acid residues onthe C-terminus of the KshA subunit and a proline on the N-terminus ofthe KshB subunit. As a result, translation of the 2A skipping peptidegenerates two individual peptides from one open reading frame.

The original eukaryotic KshAB construct was subcloned into the CMVlentiviral expression vector to generate stable Hep3B cells lines. KshABwas expressed as a cytosolic enzyme whose expression levels wereadequate for identification by anti-FLAG and anti-HA western blot (FIG.62), however, RP-HPLC analysis revealed the enzyme lacked thehydroxylase activity identified with the KshAB bacterial lysate (FIG. 63panels a & c). Recently, the crystal structure of the KshA subunit hasshown the enzyme is dependent on iron-sulfur prosthetic groups for9a-hydroxylase activity. Iron-sulfur clusters are responsible formediating the transfer of electrons in redox reactions between thesubunit and substrate. The mitochondria of eukaryotic cells are known tobe a major contributor in iron-sulfur cluster (ISC) biogenesis. Thus, wereasoned that a loss in enzyme activity was potentially a consequence ofthe bacterial Fe—S clusters failing to assemble at the active site. Weresolved this obstacle by redirecting expression of the KshAB subunitsto the mitochondria. This was accomplished by modifying the KshAB DNAconstruct with N-terminal aconitase2 mitochondrial targeting sequences(MTS) onto both A and B subunits using Gibson assembly and synthetic DNArepair strings (FIG. 61). Modifying the A and B subunit withmitochondrial targeting sequences ensured the colocalization of KshAB inan environment rich in cofactors (NADH, iron-sulfur clusters) and a moreevolutionarily conserved iron-sulfur cluster (ISC) assembly machinery.The aconitase2 MTS is a polypeptide sequence (35 amino acids) thatadopts the amphiphilic a-helical secondary structure responsible fordirecting proteins to the mitochondria. The aconitase2 MTS contains anative protease cleavage signal 31 residues within the sequence. Removalof the MTS sequence ensures the MTS residues will not interfere with thematurated enzyme once localized within the mitochondrial matrix.

We found that targeting KshAB expression to the mitochondria restoredhydroxylase activity. As proof of concept, Hep3B cells were transientlytransfected with pDest51-MTS KshAB for 48 hours and incubated with 15.7μg (10 μM) progesterone (PD) spiked with 100 nCi C4-¹⁴C labeled PD(t_(r)=13.8 min) for an additional 48 hours. RP-HPLC analysis revealedthe PD substrate was completely utilized to produce a new peak with a5.2 minute retention and a lambda max of 245 nm (FIG. 63 panels b & d).This 5.2 minute peak matched the lambda max and retention time of the9-OHPD identified in the bacterial lysate. Additionally, the 9-OHPDproduct peak contained C4-¹⁴C scintillation events, confirming the 5.2minute peak was a product of PD catabolism. In contrast, the Hep3B cellsexpressing the cytosolic form of KshAB lacked 9α-hydroxylase activity asobserved by the inability to form a 5.2 minute peak with a lambda max of245 nm (FIG. 63 panels a & c).

To determine whether the addition of the aconitase2 MTS resulted inlocalization of the subunits to the mitochondria, we transientlytransfected Hep3B cells with the mitochondrial targeted KshAB andcytosolic KshAB constructs for 48 hours. Cells were immunostained withfluorescent antibodies against the HA tag of the KshB subunit,co-stained with Mito Tracker Far-red, and then analyzed by confocalmicroscopy (FIG. 64). The merged channel show greater signalcolocalization (white) between the mitochondrial targeted KshAB andmitotracker than between cytosolic targeted KshAB and mitotracker.

With verification that the activity of KshAB was restored, MTS-KshAB wassubcloned into the plenti-CMV-Blast lentiviral expression vector togenerate stable Hep3B MTS-KshAB expressing cells lines. Stable Hep3BMTS-KshAB cells were grown to confluency in 60 mm dishes and incubatedwith 15.7 μg (10 μM) progesterone spiked with 100 nCi C4-¹⁴C PD. At theindicated time points (1, 2, 4, 6, 8, 12, 24, 36, and 48 hours), cellsand media were extracted with ethyl acetate, and the lipid profiles wereanalyzed by RP-HPLC. Spectral data and C4-¹⁴C scintillation events fromthe RPHPLC analysis of MTS-KshAB Hep3B cells revealed the9-hydroxypregn-4-ene-3,20-dione (9-OHPD; t_(r)=5.2 minutes; λ_(max) 245nm) product accumulated over the 48 hour time course (FIG. 65). Theretention time and lambda max of the 5.2 minute peak matches the 9-OHPDpeak produced with the KshAB bacterial lysate. Quantitative analysis ofboth the area under the curve (AUC) and counts under the curve (CUC)reinforces that as PD is catabolized by Hep3B MTS-KshAB cells, aconcomitant formation of 9-OHPD is observed (FIG. 66).

We next set out to characterize the activity of our MTS-KshAB expressingHep3B cells against the primed ring opening substrate,pregn-1,4-diene-3,20-dione (PDD). The PDD substrate (λ_(max) 247 nm;t_(r)=10.0 minutes) was enzymatically produced by incubation ofprogesterone (PD) with an aliquot of Δ¹-KstD from the IMAC partialpurification (FIG. 67). Following desaturation of PD by Δ¹-KstD, this3-ketosteroid product requires the hydroxylation of the C9 bond ofring-B by KshAB to form the unstable product,9-hydroxypregn-1,4-diene-3,20-dione (9-OHPDD). The B-ring of 9 OHPDD isthen subject to spontaneous non-enzymatic cleavage with concomitantaromatization of ring-A to form the product3-hydroxy-9,10-secopregn-1,3,5(10)-triene9,17-dione (3-HSP) whichdemonstrates a characteristic lambda max of 280 nm and is an indicatorthat ring opening has been achieved. We first confirmed that Hep3Bcontrol cells lacked the ability to form 3-HSP by incubatingnon-transduced cells with 15.6 μg (10 μM) of PDD for 72 hours. Following24, 48, and 72 hours incubation, cells and media were extracted withethyl acetate, and the lipid profiles were analyzed by RPHPLC. Analysisshows that Hep3B control cells lack the ability to produce 3-HSP, or anyadditional peaks with a retention time of 7.2 minutes and lambda max of280 nm (FIG. 68). To determine whether the MTS-KshAB Hep3B cells couldproduce 3-HSP from the appropriate intermediate, cells were incubatedwith 7.85 μg (5 μM) of PDD for 72 hours. We used half the amount of thePDD substrate for this experiment to observe the formation anddegradation of 3-HSP as observed with the Δ¹-KstD Hep3B incubated with9-OHPD. RP-HPLC analysis (FIG. 69 panel a) and the quantitative analysisof the area under the curve at λ 245 nm (FIG. 70 panel a) of theMTS-KshAB Hep3B cells shows the PDD substrate decrease with time. By 36hours, the PDD substrate has been depleted. Analysis at λ 280 nm revealsthat as PDD is utilized, a peak with a 7.2 minute retention time and alambda max of 280 nm accumulates (FIG. 69 panel b & 70 panel b). Anidentical peak, corresponding to 3-HSP, was observed with pregnane ringopening by Δ¹-KstD and KshAB clarified bacterial lysates followingincubation with progesterone (PD) and with Δ¹-KstD Hep3B cells incubatedwith 9-hydroxypregn-4-ene-3,20-dione (9-OHPD). Interestingly, timepoints following 36 hours shows 3-HSP decrease over the remaining 72hours (FIG. 71 panels b & 72b). As previously observed with Δ¹-KstDHep3B cells incubated with 9-OHPD, the reduction in 3-HSP suggest thatHep3B cells have endogenous metabolic capability to further modify thepregnane ring once opened. Quantitative analysis of the 3-HSP area underthe curve shows that Hep3B cells expressing MTS-KshAB not onlycatabolized the PDD substrate, but once exhausted, can also catabolizethe 3-HSP that was produced. An overview of quantitative analysis of PDDcatabolism and formation and degradation of 3 HSP over the 72 hour timecourse has been provided (FIG. 73).

Next, we verified MTS-KshAB could be independently expressed in U-937cells (FIG. 74). Stable MTS-KshAB expressing monocytes were generatedusing lentiviral

transduction. The MTS-KshAB U-937-derived macrophages were incubatedwith 15.7 μg (10 μM) progesterone (PD) spiked with 100 nCi C4-¹⁴Clabeled PD (t_(r)=13.8 min) for 72 hours. Analysis of U-937 MTS-KshABmacrophages reveal the PD substrate was exhausted by 72 hours.Concomitant to PD catabolism, 9-hydroxypregn-4-ene-3,20 dione (t_(r)=5.2min) is observed by the formation of a new peak with a retention time of5.2 minutes, a λ_(max) of 245 nm, and confirmed by C4-¹⁴C scintillationevents (FIG. 74 panels b & d). In contrast, U-937 control cells lack theability to catabolize PD to 9-OHPD, as seen by the absence of a peakwith a 5.2 minute retention time (FIG. 74 panels a & c).

To confirm these cells could generate 3-HSP when provided theappropriate intermediate, the MTS-KshAB U-937-derived macrophages wereincubated with 15.6 μg (10 μM) pregn-1,4-diene-3,20-dione (PDD,t_(r)=10.0 min) produced and isolated from partially purified Δ¹-KstD.Analysis of U-937 MTS-KshAB cells following 72 hours incubation showformation of 3-HSP (t_(r)=7.2 min) (FIG. 75 panels b & d). In contrast,control U937 cells lack the metabolic capability to produce 3-HSP due tolacking the ability to hydroxylate C9 of ring-B (FIG. 75 panels a & c).

Co-Expression of Δ¹-KstD and MTS-KshAB in Hep3B Cells

To determine whether Hep3B cells could co-express Δ¹-KstD and MTS-KshABto catalyze the formation of3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) fromprogesterone (PD), Hep3B cells were transiently transfected with equalquantities of pDest51-Δ¹-KstD and MTS-KshAB. Following 48 hours foradequate protein expression, cells were incubated with 15.7 μg (10 μM)progesterone spiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) foran additional 36 hours. RP-HPLC analysis of the 36 hour time courserevealed a robust conversion of the PD substrate to 9-OHPD (λ_(max) 245nm; t_(r)=5.2 minutes) by 6 hours. Although PDD (λ_(max) 247 nm;t_(r)=10.0 minutes) was not observed at 6 hours, analysis at λ280 nmreveals the formation of 3-HSP (FIG. 76). By 12 hours, the substrate andall intermediates had been catabolized to form 3 HSP. Interestingly,time points at 24 and 36 hours reveal that after the substrates andintermediates had been completely exhausted, 3-HSP is being furthermetabolized. The accumulation of C4-¹⁴C scintillation events at 6.5minutes confirms that once the cholestane ring has been opened it isbeing modified to have an increased polarity, as observed by a decreasein retention time.

Co-Expression of MTS-KshAB and Δ¹-KstD from a Tricistronic Vector

Following confirmation that MTS-KshAB and Δ¹-KstD could besimultaneously expressed to produce3-hydroxy-9,10-secopregn-1,3,5(10)-triene-9,20-dione (3-HSP) from thesubstrate progesterone (PD), our next step was to design and assemble atricistronic vector for co-expressing MTS-KshAB and Δ¹-KstD from asingle construct. Two vectors were designed to co-express MTS-KshAB andΔ¹-KstD by inserting a Thosea asigna 2A skipping peptide (T2A) or aPorcine teschnovirus-1 2A skipping peptide (P2A) between the two enzymes(FIG. 77). The T2A and P2A vectors were characterized by assessing KshABand Δ¹-KstD protein levels by Western blot and activity by RP-HPLCanalysis. Hep3B cells were transiently transfected with the T2A and P2Avectors. Following 48 hours to allow for adequate protein expression,cells were analyzed by Western blot and duplicate dishes were incubatedwith 15.7 μg (10 μM) progesterone spiked with 100 nCi C4-¹⁴C labeled PD(t_(r)=13.8 min) for an additional 48 hours. The Western blot resultsreveal that co-expressing MTS-KshAB and Δ¹-KstD with the T2A skippingpeptide resulted in higher expression of the Flag tagged KshA subunit,the HA tagged KshB subunit, and the Flag tagged Δ¹-KstD enzyme (FIG.78). In addition, RP-HPLC analysis revealed the T2A transfected cellshad higher enzyme activity than the P2A transfected cells. The T2Atransfected cells were able to completely catabolize the PD substrateand intermediates (FIG. 79 panel d) resulting in the formation of 3-HSP(t_(r)=7.2 min, λ_(max) 280 nm) (FIG. 79 panel e) and additionaldownstream degradation products (FIG. 79 panel f). The activity of theP2A construct was less, as seen by the presence of residualprogesterone, 9-OHPD (FIG. 79 panel a), and a lack of 3-HSP formation(FIG. 79 panels b & c). These findings suggest that the activity ofΔ¹-KstD is the rate limiting step in 3-HSP formation in the P2Aconstruct. Thus, the T2A construct was used as the standard tricistronicvector for subsequent experiments.

Co-Expression of P450-FdxR-Fdx, HSD2, MTS-KshAB, and Δ¹-KstD from aPentacistronic Vector (the Cholesterol Catabolism Cassette or CCC)

Lastly, to express enzymes required to catabolize cholesterol to 3-HSP,we modified the MTS-KshAB-T2A-Δ¹-KstD vector to co-express theP450-FdxR-Fdx fusion protein and HSD2 enzymes using repair strings, DNAfragments obtained from restriction digest, and Gibson assembly (FIG.80). Due to the increased activity observed with the T2A skippingpeptide used for co-expressing MTS-KshAB and Δ¹-KstD; we designed arepair sting to include a second T2A peptide between the HSD2 enzyme andMTS-KshA subunit. In total, this vector includes two P2A and two T2Aribosomal skipping peptides which alternate between the enzymes. Thefinal pentacistronic construct expresses all enzymes in this order:P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹⁻KstD. To characterizethe pentacistronic cassette and to confirm the enzymes were expressedand functional, Hep3B cells were transiently transfected with thecholesterol catabolizing cassette (CCC). Following 48 hours ofincubation, protein levels of the enzymes were assessed (FIG. 81) andduplicate dishes were incubated with 15.7 μg (10 μM) progesterone (PD)spiked with 100 nCi C4-¹⁴C labeled PD (t_(r)=13.8 min) for an additional24 and 72 hours (FIG. 82). Anti-Flag Western blot analysis confirmed theFlag tagged KshA and Δ¹-KstD enzymes were expressed. Additionally, theanti-HA Western blot identified the HA tagged KshB enzyme. Although theFlag tagged P450-FdxR-Fdx fusion protein and HSD2 enzyme (primaryantibody only) were not identified in these western blots, theidentification of KshAB and Δ¹-KstD supports that the enzymes are beingexpressed from the correct open reading frame (FIG. 81). RP HPLCanalysis revealed the majority of the progesterone (PD) substrate wascatabolized into downstream degradation products (FIG. 82).Interestingly, scintillation events accumulated in the solvent front(2-3 minutes), suggesting that 3-HSP was further modified once formed.Similar results were observed in the MTS-KshAB-T2A-Δ¹-KstD transfectedHep3B cells after incubation with PD for 48 hours.

Following verification that the cholesterol catabolizing cassette (CCC)was functional, U-937 cells stably expressing the CCC were generatedusing lentiviral transduction. U-937 cells (CCC and control) were platedin 60 mm dishes and differentiated into macrophages (as described inmethods). Five day old macrophages were loaded with 5 μg C4-¹⁴C labeledLDLs (18 nCi C4-¹⁴C-cholesterol) for 24 hours. Following incubation, themedia containing the radiolabeled LDLs was removed and the cells werewashed with PBS. The cells were provided with new media and cholesterolretention was monitored by measuring C4-¹⁴C scintillation events in thecells at timed intervals for 48 hours. The data from two independentexperiments with four replicates revealed more C4-¹⁴C labeledcholesterol in the control cells as compared to the CCC cell line (FIG.83). The decrease in C4-¹⁴C scintillation events in control macrophagescan likely be attributed to cholesterol efflux via the ABCA1 lipidtransporter. In theory, when cholesterol levels become low, ABCA1mediated cholesterol efflux is suppressed. However, the experimentaldesign for these experiments measure retention and at this time it isnot clear how much cholesterol leaves via ABCA1 mediated export and howmuch is degraded via catabolism in the CCC cell line.

Materials and Methods

Codon Optimization and cDNA Synthesis of the Bacterial Enzymes

1. Cholesterol Dehydrogenase (CholD)

To obtain DNA encoding humanized CholD, the amino acid sequence ofcholesterol dehydrogenase, gene: 1917_07855 from Mycobacteriumtuberculosis (strain Haarlem/NITR202), accession number: R4M4B2 wasreverse translated using GeneOptimizer software (Gene Art; Waltham,Mass. USA) set to Homo sapiens codon usage. GeneOptimizer software wasalso used to design flanking sequences that contained Gateway attachmentsites (attB1 and attB2) and restriction enzyme recognition sites (5′:MfeI and BamHI; 3′: SmaI, EcoRI, and BgIII) which were added to aid subcloning. In addition, 5′ of the CholD sequence a tobacco etch proteaserecognition site (TEV site) for cleaving upstream fusion proteins, a 6×His tag, Kozak consensus sequence, a tetracysteine tag, and a Flag tagwere added to aid purification and detection of the recombinant proteinafter expression. This humanized CholD (FIG. A1) construct was thensynthesized and inserted into the pMK-RQ vector (GeneArt). Upon arrivalthe lyophilized DNA was resuspended in H₂O at a concentration of 100ng/μL. The concentration of the DNA was measured using a BioRadSmartspec 3000 spectrophotometer.

2. Anoxic Cholesterol Metabolism Enzyme B (acmB)

To obtain DNA encoding humanized acmB, the amino acid sequence of anoxiccholesterol metabolism enzyme B(Cholest-4-en-3-one-delta1-dehydrogenase), gene: acmB fromSterolibacterium denitrificans (strain Chol-1st), accession number:A9XWD7 was reverse translated using GeneOptimizer software set to H.sapiens codon usage. GeneOptimizer software was used to design flankingsequences that contained Gateway attachment sites (attB1 and attB2) andrestriction enzyme recognition sites (5′: MfeI and BamHI; 3′: NaeI,SmaI, EcoRI, and BgIII), which were added to aid sub cloning. Inaddition, 5′ of the acmB sequence a TEV site, Kozak consensus sequenceand 3′ HA tag were added to aid in purification and detection of therecombinant protein after expression. This humanized acmB construct(FIG. A5) was then synthesized and inserted into the pMA-RQ vector(GeneArt). Upon arrival, the lyophilized DNA was resuspended in H2O at aconcentration of 100 ng/μL.

3. 3-Ketosteroid Δ¹-Dehydrogenase (Δ¹-KstD)

To obtain DNA encoding humanized Δ¹-KstD, the amino acid sequence of3-ketosteroid Δ¹-dehydrogenase, gene: KstD1 from Rhodococcuserythropolis (strain PR4/NBRC 100887), accession number: C0ZQP5 wasreverse translated using GeneOptimizer software set to H. sapiens codonusage. GeneOptimizer software was used to design flanking sequences thatcontained Gateway attachment sites (attB1 and attB2) and restrictionenzyme recognition sites (5′: MfeI and BamHI; 3′: EcoRI, and BgIII),which were added to aid sub cloning. In addition, 5′ of the Δ¹-KstDsequence a TEV site, a 6× His tag, Kozak consensus sequence,tetracysteine tag, and a Flag tag were added as discussed above. Thehumanized Δ¹-KstD construct (FIG. A15) was then synthesized and insertedinto the pUC57 vector (GenScript). Before use, the DNA was resuspendedin H₂O at a concentration of 200 ng/μL.

4. 3-Ketosteroid 9α-Hydroxylase (KshAB)

Two different 3-ketosteroid 9α-hydroxylase (KshAB) bicistronic vectorswere synthesized, one for prokaryotic and the other for eukaryoticexpression. The genes used to design the two constructs were: kshA5Bfrom Rhodococcus rhodochrous (strain DSM 43269), KshA5 gene: kshA5,accession number: F1CMY8; and KshB gene: kshB, accession number: F1CMX3.The amino acid sequence of KshAB was reverse translated usingGeneOptimizer software for Escherichia coli and Homo sapiens codonusage.

The prokaryotic KshAB vector was designed as a bicistronic construct byinserting a second Shine-Dalgarno sequence following the 3′ end of KshA.The second Shine-Dalgarno was shifted by one nucleotide to produce asecond open reading frame for coexpression of KshB. Both subunits weredesigned with 5′ cell penetrating peptides (CPPs) from the HIV-TATprotein (MGYGRKKRRQRRR; SEQ ID NO:9), short linker peptides (aminoacids: GAS), and 6× His tags. GeneOptimizer software was used to designflanking sequences that contained Gateway attachment sites (attB1 andattB2) and restriction enzyme recognition sites (5′ BamHI; 3′ PstI andan EcoRI between the A and B subunits) which were added to aid subcloning. The open reading frame was also optimized for expression in E.coli.

The eukaryotic KshAB vector was designed as a bicistronic construct byinserting the Porcine teschovirus-1 2A skipping peptide following the 3′end of KshA. In addition, a Kozak consensus sequence and Flag tag wereadded 5′ of KshA to aid in detection of the A subunit. Similarly, an HAtag was added 5′ of KshB for detection of the B subunit. Gatewayattachment sites (attB1 and attB2) and restriction enzyme recognitionsites (5′ BgIII and XbaI; 3′ BamHI and MfeI) which were added to aid subcloning. The open reading frame was also optimized for expression in H.sapiens.

The prokaryotic (FIG. A27) and eukaryotic (FIG. A31) constructs weresynthesized and inserted into pMA-RQ (GeneArt). Before use, the DNA wasresuspended in H₂O at a concentration of 100 ng/μL.

Codon Optimization and cDNA Synthesis of the Human Enzymes

P450 Side Chain Cleavage Enzyme-FerredoxinReductase-Ferredoxin-P2A-30-Hydroxysteroid Dehydrogenase 2 Construct(P450-FdxR-Fdx-P2A-HSD2 Construct)

The P450-FdxR-Fdx-P2A-HSD2 construct is a bicistronic expression vectorencoded the P450-FdxR-Fdx fusion protein and 30-hydroxysteroiddehydrogenase 2 enzyme (HSD2), separated by the 2a “ribosomal-skippingpeptide”. The three genes used in the design of the P450 fusion proteinare listed from 5′ to 3′: a P450 side chain cleavage enzyme, CYP11A,from H. sapiens, accession number: P05108; ferredoxin reductase, FDXR,from H. sapiens, accession number: P22570; and ferredoxin FDX1, from H.sapiens, accession number: P10109. Truncated versions of P450 side chaincleavage enzyme, ferredoxin reductase (FdxR), and ferredoxin (Fdx)enzymes were fused using short linkers: amino acid sequence TDGTSbetween P450 and FdxR; and amino acid sequence TDGAS between FdxR andFdx (FIG. A10). The native P450 mitochondrial targeting sequence (MTS)was retained to direct the fusion protein to the mitochondria; however,the MTS for FdxR and Fdx were omitted. A Flag tag was added to the 3′end of Fdx protein to aid identification. The P450-FdxR-Fdx-P2A-HSD2construct was designed with a Porcine teschovirus-1 2A skipping peptidefollowing the 3′ Fdx Flag tag for co-expressing the HSD2 enzyme. TheHSD2 gene used for this construct is 3β-hydroxysteroid dehydrogenase(Δ⁵⁻⁴-isomerase) from H. sapiens, gene: HSD3B2, accession number:P26439. To obtain DNA encoding the P450-FdxR-Fdx-P2A-HSD2 construct, theamino acid sequence was reverse translated using GeneOptimizer softwareset to H. sapiens codon usage. GeneOptimizer software was also used todesign flanking sequences that contained Gateway attachment sites (attL1and attL2) and restriction enzyme recognition sites (5′ BgIII and XbaI;3′ BamHI and MfeI), which were added to aid sub cloning. TheP450-FdxR-Fdx-P2A-HSD2 construct (FIG. All) was then synthesized andinserted into pMK-RQ vector (GeneArt). Before use, the DNA wasresuspended in H₂O at a concentration of 100 ng/μL.

Amplification of Initial Vectors

Aliquots of omnimax 2T1^(R) cells (50 μL) were transformed withpMK-RQ-CholD, pMA-RQ-acmB, pUC57-Δ¹-KstD, pMA-RQ-KshAB (pro),pMA-RQ-KshAB (euk), or pMK-RQ-P450-FdxR-Fdx-P2A-HSD2 vectors,respectively, by incubating bacterial cells with 1 μL of the indicatedplasmid DNA (on ice for 30 minutes). Following incubation, cells wereheat shocked for 30 seconds in a 42° C. water bath. Transformants wereplaced back on ice for 2 minutes. Then 250 μL of SOC media was added.Transformants were placed in a shaking incubator at 37° C. at 250 RPMfor 1 hour. Following incubation, 30 μL and 70 μL of transformants wereplated onto two LB agar plates containing 100 μg/mL ampicillin (pMA-RQand pUC57) or 50 μg/mL kanamycin (pMK-RQ) and grown for 14 hours at 37°C.

Isolation of Initial Vector DNA

To isolate initial vectors, twenty colonies of each were selected andplated on one 100 μg/mL ampicillin (pMA-RQ and pUC57) or 50 μg/mLkanamycin (pMK-RQ) LB agar plate and grown for 14 hours at 37° C. Threeto nine clones were selected and streaked onto additional LB agar platescontaining the same concentration of antibiotic and grown for 14 hoursat 37° C. The clones were used to inoculate 5 mL LB broth startercultures containing 100 μg/mL ampicillin (pMA-RQ and pUC57) or 50 μg/mLkanamycin (pMK-RQ). Starter cultures were grown at 250 RPM for 14 hoursat 37° C. Initial vector DNA (pMK-RQ-CholD, pMA-RQ-acmB, pUC57-Δ¹-KstD,pMA-RQKshAB (pro), pMA-RQ-KshAB (euk), or pMK-RQ-P450-FdxR-Fdx-P2A-HSD2)was isolated using the Qiagen mini kit, screened by restriction enzymedigest, and verified by DNA sequence analysis.

BP Reaction and Transformation of Omnimax 2T1R Cells withpEntr221-CholD, acmB, Δ¹-KstD, KshAB (pro), or KshAB (euk) vectors

BP reactions were assembled using equimolar concentrations (50 fmols) ofeach initial vector and pDonr221. BP Clonase II (2 μL) was added to theDNA, and the final volume was adjusted to 10 μL with TE buffer.Reactions were incubated at 25° C. for one hour. To terminate the BPreaction, 1 μL of Proteinase K was added and incubated at 37° C. for 10minutes. Omnimax 2T1^(R) cells (50 μL) were transformed with the entryvector product by incubating cells with 1 μL DNA on ice for 30 minutes.Following incubation, the cells were heat shocked for 30 seconds in a42° C. water bath. Transformants were placed back on ice for 2 minutesand 250 μL of SOC media was added. Transformants were placed in ashaking incubator at 37° C. for 1 hour at 250 RPM. Following incubation,30 μL and 70 μL of transformants were plated onto two LB agar platescontaining 50 μg/mL kanamycin and grown for 14 hours at 37° C.

Isolation of pEntr221-CholD, acmB, Δ¹-KstD, KshAB (pro), and KshAB (euk)DNA

To isolate pEntr221-CholD (FIG. A3), acmB (FIG. A7), Δ¹-KstD (FIG. A17),KshAB (pro) (FIG. A29) and KshAB (euk) (FIG. A33), twenty colonies ofeach entry clone were selected and plated on one 50 μg/mL kanamycin LBagar plate and grown for 14 hours at 37° C. Three to nine clones of eachpEntr221 vector were selected from the plate and streaked onto anadditional LB agar plate containing 50 μg/mL kanamycin and grown for 14hours at 37° C. The clones were used to inoculate 5 mL LB broth startercultures containing 50 μg/mL kanamycin and grown at 250 RPM for 14 hoursat 37° C. Entry clone vectors were isolated using the Qiagen mini kit,screened by restriction enzyme digest, and verified by DNA sequenceanalysis.

LR Reaction and Transformation of Omnimax 2T1R Cells with ExpressionConstructs

LR reactions were assembled using equimolar concentrations (50 fmols) ofeach pEntr221 clone and the desired expression vector. Prokaryoticexpression vectors included pBAD-Dest49 (CholD (FIG. A4), acmB (FIG.A8), Δ¹-KstD (FIG. A18)) and pDest14 (KshAB (pro) (FIG. A30)).Eukaryotic expression vectors included pEF-Dest51 (Δ¹-KstD (FIG. A21),KshAB (euk) (FIG. A34), and P450-FdxR-Fdx (FIG. A13)); pLenti-CMV-Blast(w706-1) (acmB (FIG. A9), Δ¹-KstD (FIG. A25), KshAB (euk) (FIG. A41),and P450-FdxR-Fdx (FIG. A14)); and pLenti-CMV-Puro (W118-1) (Δ¹-KstD(FIG. A26)). LR Clonase II (2 μL) was added to the DNA and the finalvolume was brought to 10 μL with TE buffer. Reactions were incubated at25° C. for one hour. To terminate the LR reaction, 1 μL of Proteinase Kwas added, and the reaction was incubated at 37° C. for 10 minutes.Omnimax 2T1^(R) cells (50 μL) were transformed with the expressionvector product by incubating cells with DNA (1 μL) on ice for 30minutes. Following incubation, the cells were placed in a 42° C. waterbath (heat shocked) for 30 seconds. Transformants were placed back onice for 2 minutes and 250 μL of SOC media was added. Transformants wereplaced in a shaking incubator (250 RPM) at 37° C. After 1 hour, 30 μL or70 μL aliquots were plated onto LB agar plates containing 100 μg/mLampicillin, and the bacteria were allowed to grow for 14 hours at 37° C.

Isolation of Expression Vectors

To isolate each of the expression vectors, twenty colonies were selectedand plated on one 100 μg/mL ampicillin LB agar plate and grown for 14hours at 37° C. Three to nine clones were selected from this plate andstreaked onto an additional LB agar plate containing 100 μg/mLampicillin. After 14 hours at 37° C., each of the clones were used toinoculate 5 mL LB broth starter cultures containing 100 μg/mL ampicillinand grown at 250 RPM for 14 hours at 37° C. Expression vectors wereisolated using the Qiagen mini kit, and the fidelity of each constructwas verified by restriction enzyme digest and activity screening(described below).

Transformation of Rosetta2 & C41 Expression Strains

Rosetta2 (pBAD-Dest49 vectors) or C41 (pDest14 vectors) E. coli cells(50 μL) were transformed with the expression vector (1 μL) by incubatingon ice for 30 minutes and heat shocking for 30 seconds in a 42° C. waterbath. Transformants were placed back on ice for 2 minutes and 250 μL ofSOC media was added. Transformants were placed in a shaking incubator at37° C. for 1 hour at 250 RPM before 30 μL or 70 μL aliquots were platedonto two LB agar plates containing 100 μg/mL ampicillin and 34 μg/mLchloramphenicol (Rosetta2 cells) or 100 μg/mL ampicillin only (C41cells) and grown for 14 hours at 37° C.

Rosetta2 and C41 Expression Strain Culture Preparation

Twenty colonies of each clone were selected and plated onto one LB agarplate containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol forRosetta2 cells or 100 μg/mL ampicillin for C41 cells. After 14 hours at37° C., one colony was selected from this plate, streaked onto anadditional LB agar plate containing the same concentration of antibioticand grown for 14 hours at 37° C. A single colony from each clone wasselected and used to inoculate 5 mL LB broth starter cultures containing100 μg/mL ampicillin and 34 μg/mL chloramphenicol (Rosetta2 cells) or100 μg/mL ampicillin only (C41 cells). Cultures were grown for 14 hoursat 37° C. shaking at 250 RPM. A 1:250 dilution of the starter culturewas used to initiate a 500 mL LB broth liquid culture containing 100μg/mL ampicillin and 34 μg/mL chloramphenicol (Rosetta2 cells) or 100μg/mL ampicillin only (C41 cells). Cultures were incubated at 37° C. forsix hours shaking at 250 rpm. Once the culture reached an OD₆₀₀ of 0.4,0.10% arabinose (pBAD-Dest49 vectors) or 300 μM IPTG (pDest14 vector)was added to induce heterologous expression. Cultures were grown at 25°C. for 24 hours shaking at 250 rpm until reaching an OD₆₀₀ of 4.0.

Bacterial Lysis

The bacterial culture was subjected to centrifugation at 4,000×g for 20minutes at 4° C. in a Sorvall Instruments RC5C using a GSA rotor. Thebacterial pellet was weighed and suspended in four volumes of chilledlysis buffer (1:4; w/v). Lysis buffer consisted of 25 mM Tris-HCl, pH7.5, 500 mM NaCl, 1 mM MgCl₂, 1 mM PMSF, 1× Calbiochem ProteaseInhibitor Cocktail Set 1, and 525U of Pierce Universal Nuclease. Forprotein purification by IMAC, 20 mM imidazole was added to this buffer.Cells were lysed using a chilled French press with a high pressuresetting of 18,000 psi with a Thermo IEC French Press Cell Disruptor. Thebacterial pellet was placed on ice and then run through the French pressa second time for adequate lysis of bacteria. The crude lysate wassubjected to centrifugation at 28,500×g for 1 hr at 4° C., to separatethe soluble and insoluble fractions, in a Sorvall Instruments RC5C withan SS34 rotor.

Clarified Lysate Activity Assessment by RP-HPLC

Independent assays were conducted with clarified lysate from CholD,acmB, Δ¹-KstD, KshAB, or empty vector transformed E. coli. For eachassay, 100 μL of clarified lysate was mixed with 100 μM substrate (3.87μg cholesterol (CL) spiked with 20 nCi ¹⁴C radiolabeled CL, 3.87 μgcholestenone (CN), 3.16 μg pregnenolone (PL), or 3.14 μg progesterone(PD) spiked with 20 nCi ¹⁴C radiolabeled PD) in an 2 mL glass HPLC vialfor 24 hours on a rotator. Following incubation, steroids were isolatedand analyzed by RP-HPLC as described below.

Δ¹-KstD Partial Purification by Immobilized Metal AffinityChromatography

Clarified lysate (23.75 mL) containing Δ¹-KstD was loaded onto anickleSepharose column (GE HiTrap Chelating HP columns, 1.6×2.5 cm).Isolation was performed by washing out unbound protein with twenty-twocolumn volumes 98:2 buffer A:B; (buffer A; 25 mM Tris-HCl, pH 7.5, and500 mM NaCl; buffer B; 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 1 Mimidazole). Δ¹-KstD was then eluted with a 2 mL/min imidazole lineargradient, collected in 9 mL fractions at 4° C. using an AKTA FPLC System(GE Healthcare). Elution occurred in two steps. Step one was 95:5solvent A:B and was followed by a linear gradient to 80:20 solvent A:Bover ten column volumes. The 80:20 solvent A:B was held for five columnvolumes before returning to 98:2 solvent A:B for two column volumes. TheN-terminal HP-Thioredoxin Δ¹-KstD fusion protein was eluted in 25 mMTris-HCl, pH 7.5, containing 500 mM NaCl and 120 mM imidazole. Elutionfraction 21 was assessed to contain a high yield of Δ¹-KstD withrelatively low contaminating proteins. Protein concentration wasdetermined using the Bio-Rad Protein assay using bovine serum albumin asa standard. The SDS-PAGE of elution fraction 21 was coomassie stainedwas analyzed with ImageJ and estimated to contain 0.385 mg/mL of Δ¹-KstDat 79.6% purity by densitometry.

Nitrotetrazolium Blue Δ¹-KstD Activity Assay

Equal volumes (5 μL) of representative fractions from the Δ¹-KstD IMACpurification were loaded onto a native gel (10% acrylamide) and ran at50 v for 5 hours at 4° C. Following electrophoresis, the gel wasincubated in 10 mL of nitrotetrazolium blue solution (160 nM phenazinemethylsulfate, 80 nM nitrotetrazolium blue, and 1.5 nM progesterone in66.7 mM Tris) for 5 minutes at 25° C.

Partially Purified Δ¹-KstD SDS-PAGE and Western Blot

To assess the purity and yield of Δ¹-KstD from the IMAC purification,aliquots of each fraction were diluted (1:1; v/v) in laemmli samplebuffer. Equal volumes (5 μL) from each fraction were separated usingSDS-PAGE on a 10% polyacrylamide gel at 50 v for 0.5 hours followed by125 v for 1.4 hours at 4° C. Protein was transferred to a PVDF membraneusing 300 mA for 2.3 hours, and probed with anti-FLAG (1:1000; SigmaF3163, from mouse). ECL anti-mouse IgG secondary antibody conjugated tohorseradish peroxidase (HRP) linked whole antibody (1:10,000, GEHealthcare NA931VS, from sheep) and SuperSignal West Femto Substrate wasused for detection of the N-terminal FLAG tag of Δ¹-KstD. DuplicateSDS-PAGE gels were coomassie stained to assess purity and yield.

Δ¹-KstD Enzyme Titration Curves

Fluorometric assays using resazurin were performed in 25 mM Tris-HCl, pH7.5 at 37° C. in a 96-well format using a BioTek Synergy 2 plate readerwith excitation 540±25 nm and emission 620±40 nm. Several concentrationsof Δ¹-KstD (0.05, 0.19, 0.37, 0.55, 1.1, 1.6, 2.12 nM) and 0.1 mg/mL BSAwere dispensed with a PD syringe. Reactions were initiated by theaddition of 20 μM resazurin and 20 μM progesterone. Measurements weremade with an N of 1 with three replicates and three blanks for baselinesubtraction by measuring the fluorescence of each well every 17 secondsfor a total of 3.5 minutes.

Δ¹-KstD Steady State Kinetic Analysis with Resazurin Assay

Initial velocities were measured by monitoring the reduction ofresazurin at 37° C. Reaction mixtures containing 0.55 nM, 1.1 nM, or 1.6nM Δ¹-KstD and 0.1 mg/mL BSA were dispensed with a PD syringe prior toinitiating the reaction by the addition of increasing concentrations ofprogesterone (1, 2.5, 5, 10, 20, 30, and 40 μM) and 20 μM resazurin in25 mM Tris-HCl, pH 7.5. Measurements were made with an N of 1 with eightreplicates and four blanks for baseline subtraction by measuring thefluorescence of each well every 17 seconds for a total of 10 minutes.

Δ¹-KstD Substrate Specificity with Resazurin Assay

Substrate specificity assays were performed in 300 μL 25 mM Tris-HCl, pH7.5. Relative fluorescent intensity was measured following 10 minutes ofincubation with several steroid substrates at 37° C. Reaction mixturescontaining 5.35 nMΔ¹-KstD and 0.1 mg/mL BSA (dispensed with a PDsyringe) were equilibrated for 30 sec before the reaction was initiatedby adding 20 μM resazurin and 20 μM of the steroid substrate. Screenedsteroid substrates include: 33-hydroxypregn-5-en-20-one (Pregnenolone)(Sigma), Pregn-4-ene-3,20-dione (Progesterone) (Sigma),(11β)-11,17,21-trihydroxypregna-1,4-diene-3,20-dione (Prednisolone)(Sigma), 4-pregnen-17-ol-3,20-dione (17-hydroxyprogesterone) (SteraloidsQ3360), 4-pregnen-21-ol-3,20-dione (11-deoxycorticosterone) (SteraloidsQ3460), (11β)-11,21-dihydroxypregn-4-ene-3,20-dione (Corticosterone)(Sigma C-2505), (11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione(Hydrocortisone) (Sigma No. H-4001),17α,21-dihydroxy-4-pregnene-3,11,20-trione (Cortisone) (Sigma C-2755)1β,21-dihydroxy-3,20-dioxopregn-4-en-18-al (Aldosterone) (Acros Organics215360050), 11β,17α,21-trihydroxy-4-pregnene-3,20-dione 21-hemisuccinatesodium salt (Hydrocortisone 21-hemisuccinate) (Sigma),5α-androstan-3α-ol-17-one (Androsterone) (Steraloids A2420),5-androsten-33-ol-17-one (DHEA/Dehydroepiandrosterone) (SteraloidsA8500), 5α-androstan-17β-ol-3-one (5a-DHT) (Steraloids A2570),4-androsten-17β-ol-3-one (Testosterone) (Steraloids A6950),4-androsten-3,17-dione (Androstenedione) (Steraloids A6030),17β-hydroxy-4-androsten-3-one 17-enanthate (Testosterone enanthate orDelatestryl) (Sigma), 3β-hydroxy-5-cholestene (Cholesterol) (Sigma),5-Cholesten-3-one (Cholestenone) (Sigma),11β-(4-dimethylamino)phenyl-170-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one(Mifepristone) (Roussel UCLAF 7A 4087 RU 38486),7α-acetylthio-3-oxo-17α-pregn-4-ene-21,17-carbolactone (Spironolactone)(Sigma), and 4-Cholesten-73-ol-3-one (7β-hydroxycholestenone)(Steraloids C6230-000). Measurements were made with an N of 1 with fourreplicates and four blanks for baseline subtraction.

Partially Purified Δ¹-KstD Reactions

Reactions containing 770 ng isolated Δ¹-KstD in 25 mM Tris-HCl, pH 7.5,200 μM resazurin, and 0.1 mg/mL BSA were incubated with 6.29 μg (100 μM)progesterone for 4 hours in a 37° C. water bath. Steroid isolation andanalysis are described below.

Gibson Assemblies Δ¹-KstD Kozak Consensus Sequence Repair

The Δ¹-KstD construct's Kozak consensus sequence was repaired bydigesting pDest51-Δ¹-KstD with BbvCI and SpeI. The BbvCI and SpeI digestremoved the TEV site, 6× His tag, Kozak consensus sequence,tetracysteine tag, Flag tag, and the first 13 nt of Δ¹-KstD'sN-terminus. The pDest51-Δ¹-KstD backbone was isolated by agarose gelelectrophoresis (0.8%), extracted from the gel using a Qiagen gelextraction kit, and the concentration of DNA was determined using ananodrop spectrophotometer. The repair string (FIG. A19) (504 nt) wasdesigned to insert a new attB1 site, Kozak consensus sequence, Flag tag,and the first 13 nt of Δ¹-KstD's N-terminus that was removed from theBbvCI and SpeI digest. The repair string was synthesized by GeneArt. Therepair string included 40 bp homology arms starting from the 3′ overhangof the BbvCI restriction site in pDest51-Δ¹-KstD (left homology arm) and40 bp homology starting from the 3′ overhang of the SpeI restrictionsite (right homology arm). The repair string (150 ng) and the linearizedpDest51-Δ¹-KstD backbone (50 ng) were assembled using Gibson Assembly(following standard procedures). The assembled vector (2 μL) was dilutedwith water (4 μL) and propagated by transforming (2 μL DNA) Omnimax2T1^(R) E. coli. Plasmid DNA from six bacterial colonies was isolatedusing the Qiagen mini kit, screened by restriction enzyme digest, andverified by DNA sequence analysis. The pDest51-Repaired Δ¹-KstDconstruct (FIG. A21) was subcloned back into the pEntr221 (FIG. A23)entry vector and the further subcloned into pBAD-Dest49 (FIG. A24),pLentiCMV-puro (w118-1) (FIG. A25) and pLenti-CMV-Blast (w706-1) (FIG.A26) expression vectors using Gateway recombination as describedpreviously.

Aconitase2 Mitochondrial Targeting Sequence Addition to KshAB(MTS-KshAB)

The KshA and KshB subunits were modified by the addition of N-terminalmitochondrial targeting sequences (MTS) from the H. sapiens Aconitase2enzyme. KshAB was modified by first linearizing pEntr221-KshAB (euk)(FIG. A33) with NaeI, a restriction enzyme with two recognition sites.The pEntr221-KshAB backbone was isolated by agarose gel electrophoresis(0.8%), extracted from the gel using a Qiagen gel extraction kit, andthe concentration of DNA was determined using a nanodropspectrophotometer. For repair string synthesis, the 35 amino acidAconitase2 protein sequence (MAPYSLLVTR LQKALGVRQY HVASVLCQRA KVAMS) wascodon optimized for H. sapiens expression by GeneArts codon optimizationsoftware and then fused to the 5′ ends of both the KshA and KshBsubunits. Due to synthesis problems of a single repair string encodingthe entire repair (likely due to the highly similar MTSs), two repairstrings were synthesized by GeneArt. The first repair string (FIG. A35)(1000 nt) encoded the Kozak consensus sequence, Aconitase2 MTS, Flagtag, and first 835 nt of KshA. The second repair string (FIG. A37) (600nt) encoded the remaining 347 nt of KshA, the Porcine Teschovirus 2Askipping peptide, and the N-terminal segment of KshB that was removed byrestriction digest. The first repair string included 40 bp homology armsstarting from the 3′ overhang of the upstream NaeI restriction site inpEntr221-KshAB (left homology arm) and 40 bp homology starting from the3′ overhang of the second repair string (right homology arm). The secondrepair string included 40 bp homology starting from the 3′ overhang ofthe first repair string (left homology arm) and 40 bp homology startingfrom the 3′ overhang of the downstream NaeI restriction site inpEntr221-KshAB (right homology arm). The repair strings (150 ng) and thelinearized pEntr221-KshAB backbone (50 ng) were assembled using GibsonAssembly (following standard procedures). The assembled vectors (2 μLeach) were diluted with water (4 μL) and propagated by transforming (2μL DNA) Onimax 2T1^(R) E. coli. Plasmid DNA from six bacterial colonieswas isolated using the Qiagen mini kit, screened by restriction enzymedigest, and verified by DNA sequence analysis. The pEntr221-MTS-KshAB(FIG. A38) construct was subcloned into the pDest51 (FIG. A40) andpLenti-CMV-Blast (w706-1) (FIG. A41) expression vectors using Gatewayrecombination as described previously.

KshAB-T2A-Δ¹-KstD and KshAB-P2A-Δ¹-KstD Tricistronic Vectors

The KshAB-T2A-Δ¹-KstD and KshAB-P2A-Δ¹-KstD construct were assembled byfirst linearizing pEntr221-MTS-KshAB (FIG. A38) with MfeI. Second, theΔ¹-KstD fragment was generated by digesting pEntr221-Repaired Δ¹-KstD(FIG. A23) with EcoRI, a restriction enzyme with two recognition sitesflanking Δ¹-KstD. Both the pEntr221-MTS-KshAB backbone and the Δ¹-KstDinsert were isolated by agarose gel electrophoresis (0.8%), extractedfrom the gel using a Qiagen gel extraction kit, and the concentration ofDNA was determined using a nanodrop spectrophotometer. The isolatedΔ¹-KstD fragment was ligated into the linearized pEntr221-KshAB backbonevector using standard ligation with NEB T4 ligase. Following ligationand transformation of the construct into Omnimax 2T1^(R) E. coli cells,the vector was sequence verified for proper insertion and orientation ofthe Δ¹-KstD insert. The ligated pEntr221-MTS-KshAB Δ¹-KstD vector waslinearized by digesting with BbvCI and SpeI to remove the KshBC-terminal stop codon and Δ¹-KstD kozak sequence. The linearizedpEntr221 MTS-KshAB Δ¹-KstD backbone was isolated by agarose gelelectrophoresis (0.8%), extracted from the gel using a Qiagen gelextraction kit, and the concentration of DNA was determined using ananodrop spectrophotometer. Two repair strings were designed with 40 bphomology arms starting from the 3′ overhangs generated from the BbvCIand SpeI digest. The repair strings were synthesized by GeneArt toencode for the KshB C-terminus (excluding the native KshB stop codon),one of two viral 2A ribosomal skipping peptides, and the N-terminus ofΔ¹-KstD that was removed from the BbvCI and SpeI digest. Forco-expressing Δ¹-KstD along with KshAB, the first repair string (FIG.42) (656 nt) was synthesized with a Thosea asigna 2A skipping peptide(T2A peptide) and the second repair string (FIG. A44) (659 nt) wassynthesized with a Porcine teschovirus 2A skipping peptide (P2Apeptide). Following the P2A and T2A skipping peptide sequences are theΔ¹-KstD Flag tag and the remaining Δ¹-KstD coding sequence removed bythe BbvCI and SpeI digest. The repair strings (150 ng) and thelinearized pEntr221 MTS-KshAB Δ¹-KstD ligated product (50 ng) wereassembled using Gibson Assembly (following standard procedure) toproduce a circularized tricistronic vector. The assembled vectors (2 μLeach) were diluted with water (4 μL) and propagated by transforming (2μL DNA each) Onimax 2T1R E. coli. Plasmid DNA from six bacterialcolonies was isolated using the Qiagen mini kit, screened by restrictionenzyme digest, and verified by DNA sequence analysis. ThepEntr221-KshAB-T2A-Δ¹-KstD (FIG. A46) and pEntr221-KshAB-P2A-Δ¹-KstDconstructs were subcloned into the pDest51 (FIG. A48) andpLenti-CMV-puro (w118-1) (FIG. A49) expression vectors using Gatewayrecombination as described previously.

P450-FdxR-Fdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD PentacistronicVector (Cholesterol Catabolizing Cassette or the CCC)

The P450-FdxR-Fdx-P2A-HSD2-T2A-KshAB-T2A-Δ¹-KstD (the CCC) (FIG. A56)construct was assembled by linearizing pEntr221-KshAB-T2A-Δ¹-KstD withNcoI. The P450-FdxR-Fdx-P2A-HSD2 fragment (FIG. A52) (3221 nt) wasgenerated by digesting pMK-RQ-P450-FdxR-Fdx-P2A-HSD2 (FIG. All) withScaI and EcoRV. The linearized pEntr221-KshAB-T2A-Δ¹-KstD (FIG. A46)backbone and P450-FdxR-FdxP2A-HSD2 fragment (FIG. A52) were isolated byagarose gel electrophoresis (0.8%), extracted from the gel using aQiagen gel extraction kit, and the concentration of DNA was determinedusing a nanodrop spectrophotometer. Two repair strings were designedwith 40 bp homology arms starting from the 3′ overhangs of thepEntr221-KshAB-T2AΔ¹-KstD vector backbone and the P450-FdxR-Fdx-P2A-HSD2fragment. The first repair string (FIG. A50) included 40 bp of homologystarting from the 3′ overhangs of the NcoI restriction site inpEntr221-KshAB-T2A-Δ¹-KstD (left homology arm) and the ScaI restrictionsite of the P450-FdxR-Fdx-P2A-HSD2 fragment (right homology arm). Thesecond repair string (FIG. A54) included 40 bp of homology starting fromthe 3′ overhangs of the EcoRV restriction site of theP450-FdxR-Fdx-P2A-HSD2 fragment (left homology arm) and the NcoIrestriction site in pEntr221-KshAB-T2A-Δ¹-KstD (right homology arm). Thefirst repair string (474 nt) includes the KshAB Kozak consensus sequenceand the P450 sequence that was lost after generating the P450-FdxR-Fdxfragment. The second repair string (1079 nt) included the C-terminal endof HSD2 excluding the native stop codon and a Thosea asigna 2A skippingpeptide (T2A peptide). The two repair strings (150 ng each), theP450-FdxR-Fdx-P2A-HSD2 digested fragment (50 ng), and the linearizedpEntr221-KshAB-T2A-Δ¹-KstD backbone (50 ng) were assembled using GibsonAssembly (following standard procedure) to produce a circularizedpentacistronic vector. The assembled vector (2 μL) was diluted withwater (4 μL) and propagated by transforming (2 μL DNA) Onimax 2T1^(R) E.coli. Plasmid DNA from six bacterial colonies was isolated using theQiagen mini kit, screened by restriction enzyme digest, and verified byDNA sequence analysis. ThepEntr221-P450-FdxR-FdxP2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD construct(FIG. A56) was subcloned into the pDest51 (FIG. A58) and pLenti-CMV-puro(w118-1) (FIG. A59) expression vectors using Gateway recombination asdescribed previously.

Mammalian Cell Culture

Hep3B (ATCC HB-8064) and HEK293FT (Thermo Fisher R70007) cells werecultured in media A: DMEM containing 1 mM sodium pyruvate, 0.5×NEAA, and10% fetal bovine serum. For experiments, Hep3B cells were maintained inmedia B: DMEM containing 1 mM sodium pyruvate, 0.5×NEAA, and 0% fetalbovine serum. U-937 monocytes (ATCC CRL-1593.2) were cultured in mediaC: RPMI-1640 media supplemented with 1×NEAA, 100 units/mL penicillin,100 μg streptomycin, and 10% fetal bovine serum. For experiments, U-937derived macrophages were maintained in media D: RPMI-1640 mediasupplemented with 1× NEAA, 100 units/mL penicillin, 100 μg streptomycin,and 2% fetal bovine serum. Mammalian cells were cultured in corning T75flasks at 37° C. and 5% CO₂.

U-937 Derived Macrophages

U-937 monocytes were differentiated into macrophages by incubation withphorbol 12-myristate 13-acetate (PMA P1585 Sigma) in media C. PMA wasprepared by diluting 100 μg PMA with 1 mL DMSO. U-937 monocytes wereseeded into 35 mm (5×10⁵ cells), 60 mm (1.5×10⁶ cells), or 100 mm dishes(6×10⁶ cells) coated with 0.1% gelatin. PMA was added to a finalconcentration of 200 nM (120 ng/mL of media) for 48 hours. Following PMAtreatment, media was removed, cells rinsed with PBS (2×), and cells wereallowed to continue differentiating in media C for 72 hours.

Transient Expression Experiments in Hep3B Cells

Plasmid DNA (2.5 μg of pDest51-MTS-KshAB or pDest51-Δ¹-KstD) was dilutedwith 250 μL (2.5 μg DNA for single enzyme expression) or 500 μL (5.0 μgDNA for dual enzyme expression) Opti-MEM in a glass vial (1 μg plasmidDNA/100 μL OptiMEM). DNA was mixed gently by tapping bottom of vial 30times. A 3:1 ratio of XtremeGene 9 (XG9) to DNA (7.5 μL XG9 for 2.5 μgDNA and 15.0 μL XG9 for 5.0 μg DNA) was added to the glass vial andmixed gently by tapping 30 times. The DNA and transfection reagent mixwere allowed to incubate at room temperature for 30 minutes. Followingincubation, the DNA/transfection reagent was added drop wise to Hep3Bcells that were pre-seeded (2.3×10⁵ cells) and grown to 90% confluencyin 60 mm dishes. Cells were allowed to incubate with transfectionreagent for 48 hours prior to analysis. For western blot analysis, cellswere washed twice with PBS and scrapped in 500 μL RIPA buffer. Foractivity assessment, the media was removed and the cells washed twicewith PBS. New media (media B) and 10 μM of the steroid substrate wereadded to cells and incubated at 37° C. At the indicated time points,cells were scrapped and removed with the media, extracted, and analyzedby RP-HPLC as described below.

Lentiviral Packaging

Lentiviral particles encoding the enzyme constructs were produced withHEK293FT cells using the third generation lentiviral packaging system(Addgene). Packaging vectors (7.5 μg PMDL, 3.75 μg RSV-REV, and 4.5 μgPMD2.G) and each transfer vector (3 μg pLenti-CMV-Blast (706-1) orpLenti-CMV-Puro (w118-1)) were diluted with 1.875 mL Opti-MEM (1 μgplasmid DNA/100 μL Opti-MEM) in a glass vial. DNA was mixed gently bytapping bottom of vial 30 times. For transfections using XtremeGene HP,a 2:1 ratio of XtremeGene HP to DNA (37.5 μL XtremeGene HP) was used.For transfections using XtremeGene 9, a 3:1 ratio of XtremeGene 9 to DNA(56.25 μL XtremeGene 9) was used. The transfection reagent was added tothe glass vial and mixed gently by tapping 30 times. The DNA andtransfection reagent mix were allowed to incubate at room temperaturefor 30 minutes. Following incubation, the DNA/transfection reagent wasadded to 12 mL of media A and mixed by inversion. The media containingthe DNA/transfection reagent was added slowly to the side of the 0.10%gelatin coated T75 flask containing HEK293FT cells that were pre-seeded(2×10⁶ cells) and grown to 70% confluency. The flask was slowly laidflat to minimize disturbing the monolayer of cells. Cells were allowedto produce lentiviral particles for 48 hours. Following incubation, theviral supernatant was removed and subjected to centrifugation (1,625×g)for 2 minutes. Next, the supernatant was syringe filtered with a 0.45 μmpolyethersulfone membrane and separated into 500 μL aliquots. Lastly thealiquots were flash frozen in a dry ice/ethanol bath and stored at −80°C.

Stable Expression of Δ¹-KstD, KshAB, P450-FdxR-Fdx-P2A-HSD2, andP450-FdxRFdx-P2A-HSD2-T2A-KshA-P2A-KshB-T2A-Δ¹-KstD in Hep3B and U-937Cells

Hep3B cells (2.3×10⁵ cells) were grown to 70% confluency in 60 mm dishesand U-937 monocytes (1.0×10⁵ cells/mL) were seeded in T25 flaskscontaining 5 mL media. Both Hep3B and U-937 cells were transduced with0.5 mL of the total 13 mL of the viral supernatant. Cells were allowedto incubate with the viral supernatant for 48 hours prior to selectionwith 0.05 mg/mL hygromycin (pLenti-PGK-Hygro (w530-1)), 0.001 mg/mLpuromycin (pLenti-CMV-Puro (w118-1)), or 0.012 mg/mL blasticidin(pLenti-CMVBlast (706-1)) antibiotic for two weeks prior to time courseexperiments.

SDS-PAGE and Western Blot of Eukaryotic Cell Lines

Cells were grown in 60 mm (Hep3B) or 100 mm (U-937 derived macrophages)dishes, washed with PBS (2×), and collected by scraping in 500 μL RIPAbuffer. Cells were mechanically lysed on ice using a syringe with a 27gauge needle. Protein samples were mixed with an equal volume of 2×Laemmli sample buffer, boiled for 5 minutes, and subjected tocentrifugation at 15,000×g for 10 minutes at 4° C. Protein samples (25μg) were separated using SDS-PAGE on a 10% polyacrylamide gel,transferred to a PVDF membrane, and probed with anti-FLAG (1:1000;Millipore MAB3118, from mouse), anti-HA (1:3000; Sigma H9658, frommouse), or anti-HSD3B2 (1:1000, Abcam ab80500, from rabbit). ECLanti-mouse IgG secondary antibody conjugated to HRP (1:10,000 GEHealthcare NA931VS, from sheep) or ECL anti-rabbit IgG (1:10,000, GEHealthcare UK Limited NA934V, from donkey) and SuperSignal West FemtoSubstrate were used for detection.

Enzyme Activity Assessment of Stable Hep3B and U-937 Cell Lines

Hep3B cells stably expressing enzyme constructs were seeded (2.3×10⁵cells) into 60 mm dishes and grown to confluency in media A. At time 0,the media was removed and the cells were washed twice with PBS. Newmedia (media B) and 10 μM of the steroid substrate were added to cellsand incubated at 37° C. U-937 derived macrophages in 100 mm dishescoated with 0.1% gelatin were prepared as previously described. Five dayold macrophages were given 5 mL media D and 10 μM of the steroidsubstrate or 50 μg C4-¹⁴C cholesterol labeled LDLs (163 nCiC4-¹⁴C-cholesterol). At the indicated time points, the cells werescraped and cells with media were removed from the dish. The steroidsanalytes were extracted and analyzed by RP-HPLC as described below.

Pregn-1,4-diene-3,20-dione (PDD) and 9-hydroxypregn-4-ene-3,20-dione(9-OHPD) Production and Isolation

To produce and isolate the pregn-1,4-diene-3,20-dione (PDD) substrate, areaction was assembled by adding 200 μL of elution fraction 20 from theΔ¹-KstD IMAC isolation, 629 μg (100 μM) progesterone (PD), 200 μMresazurin, 10 μg/ml BSA, and the final volume brought to 20 mL withTris-HCL pH 7.5. Following 24 hours of incubation at 25° C. on arotator, the reaction was stopped and extracted using ethyl acetate(2:1; v/v), twice. The ethyl acetate was dried using nitrogen gas, andsteroid analytes resuspended in 500 μL EtOH. The bioconversion analyteswere analyzed as described below. RPHPLC analysis revealed a 95%conversion of progesterone (PD) to pregn-1,4-diene-3,20 dione (PDD).Based on the progesterone standard curve, the final concentration of thePDD stock was 2.54 mM.

To produce and isolate the 9-hydroxypregn-4-ene-3,20-dione (9-OHPD)substrate, a reaction was assembled by adding 7 mL of the bacterialKshAB clarified lysate, 1.25 mg progesterone, and 70 μM NADH. Following48 hours of incubation at 25° C. on a rotator, the reaction was stoppedand extracted using ethyl acetate (2:1; v/v), thrice. The steroidanalytes were dried using nitrogen gas, and resuspended in 500 μL EtOH.The bioconversion analytes were analyzed as described below. RP-HPLCanalysis revealed a 76.6% conversion of progesterone (PD) to9-hydroxypregn-4-ene-3,20-dione (9-OHPD). Based on the progesteronestandard curve, the final concentration of the 9-OHPD stock was 5.8 mM.

C4-¹⁴C-Progesterone and C4-¹⁴C-Cholesterol Stocks (10 mM)

Stocks (10 mM at 20 nCi/μL) of C4-¹⁴C-Progesterone were prepared bydiluting 20 μL C4-¹⁴C-Progesterone (ARC 1398A, Progesterone [4-13C],S.A. 55 mCi/mmol, 50 μCi/vial) with 50 μL of 20 mM unlabeledprogesterone (in EtOH) and 30 mL EtOH. Stocks (10 mM at 20 nCi/μL) ofC4-¹⁴C-cholesterol were prepared by diluting 50 μL C4-¹⁴C-cholesterol(Perkin Elmer 250 μCi [9.25 mBq] 50.8 mCi/mmol [1.88 Gbq/mmol] in 6.25mL EtOH) with 50 μL of 20 mM unlabeled cholesterol (in EtOH).

C4-¹⁴C-Cholesterol LDL Labeling

C4-¹⁴C-cholesterol (Perkin Elmer 250 μCi [9.25 mBq] 50.8 mCi/mmol [1.88Gbq/mmol] in 6.25 mL EtOH) was used to radiolabel human low densitylipoproteins (Alfa Aesar J65039 [BT-903] 5 mg/mL). First, 4.0 μCi ofC4-¹⁴C-cholesterol (60.8 μg in 200 μL EtOH) was added to a 2 mL glassvial. To reduce the volume of EtOH the C4-¹⁴C-cholesterol was initiallysuspended in, the initial volume was dried down to approximately 20 μL.Next, a 250 μL aliquot containing 1.25 mg of human low densitylipoproteins was added to the 4.0 μCi of C4-¹⁴C-cholesterol (60.8 μg).The C4-¹⁴C cholesterol was partitioned into the LDLs by placing theglass vial in an ultrasonic water bath for 10 minutes followed byincubation at room temperature on a rotator for three days. The finalconcentration of the C4-¹⁴C-cholesterol labeled LDL stock was 4.6 μg/μLLDL labeled with 15 nCi/μL (225.19 ng/μL) C4-¹⁴C-cholesterol.

C2,3,4-¹³C3-Cholesterol LDL Labeling

C2,3,4-¹³C3-cholesterol (Cambridge Isotopes, CLM-9139-0.002) was used toradiolabel human low density lipoproteins (Alfa Aesar J65039 [BT-903] 5mg/mL). First 2 mg of C2,3,4-¹³C3-cholesterol was resuspended in 517.26uL EtOH to prepare a 10 mM stock. From this 10 mM stock ofC2,3,4-¹³C3-cholesterol, 60.75 μg (in 15.7 μL EtOH) was added to a 2 mLglass vial. Next, a 250 μL aliquot containing 1.25 mg of human lowdensity lipoproteins was added to the 60.75 μg C2,3,4-¹³C3-cholesterol.The C2,3,4 ¹³C3-cholesterol was partitioned into the LDLs by placing theglass vial in an ultrasonic water bath for 10 minutes followed byincubation at room temperature on a rotator for three days. The finalconcentration of the C2,3,4-¹³C3-cholesterol labeled LDL stock was 4.7μg/μL LDL labeled with 228.64 ng/μL C2,3,4-¹³C3-cholesterol.

U-937-Derived Macrophage LDL Loading for C4-¹⁴C-Cholesterol EffluxAnalysis

Five day old U-937-derived macrophages (prepared in 60 mm dishes aspreviously described) were incubated with 5 μg C4-¹⁴C labeled LDLs (18nCi C4-¹⁴C-cholesterol) for 24 hours. Following incubation with C4-¹⁴Clabeled LDLs, the media was removed, cells were washed with PBS (2×),and new media (media D) was given for an additional two days. At therespective time points, media was removed, cells were washed with PBS(2×), and scraped in 500 μL RIPA buffer. Scintillation events from themedia (250 μL of the 2 mL) and cells (250 μL of 500 μL) were suspendedin 4 mL of Beckman Ready Safe scintillation fluid, mixed by vortexting,and analyzed with a Beckman LS 6500 multi-purpose scintillation counterfor 10 minutes with luminex correction enabled. Scintillation eventswere normalized to total protein. Measurements were made with an N of 2in quadruplicate.

U-937 Derived Macrophage LDL Loading for C2,3,4-¹³C3-Cholesterol LC-MSAnalysis

Five day old macrophages (prepared in 100 mm dishes as previouslydescribed) were incubated with 50 μg C2,3,4-¹³C3-cholesterol labeledLDLs (159.84 nCi C4-¹⁴C-cholesterol) for 72 hours. Following incubationwith C2,3,4-¹³C3-cholesterol labeled LDLs, cells were scraped andremoved with the media. Cells and media were extracted with ethylacetate (2:1; v/v), twice. The ethyl acetate was dried under nitrogengas and analyzed by LC-MS as described below.

BODIPY 493/503 Staining and Confocal Microscopy

U-937 derived macrophages were prepared as previously described in 35 mmglass bottom dishes. Following five days of differentiation, macrophageswere given 50 μg/mL LDL (Alfa Aesar J65039 [BT-903] 5 mg/mL) oracetylated LDL ((Alfa Aesar J65029 [BT-906] 2.5 mg/mL) for 24 hours inmedia D. Following incubation with LDLs, cells were washed twice withPBS and stained with 20 μg/mL BODIPY 493/503 in PBS for 30 minutes at37° C. BODIPY 493/503(Difluoro{2-[1-3,5-dimethyl-2H-pyrrol-2-ylidene-N)ethyl]-3,5-dimethyl-1H-pyrrolato-N}boron)was prepared in DMF at a stock concentration of 2 μg/ml. Followingstaining, cells were washed with PBS (2×) and given 2 mL media D.Imaging was performed with a Nikon A1 Confocal Microscope using anexcitation of 488 nm and emission of 525/50 nm at 60× magnification.

Steroid Isolation

Reactions were extracted twice with ethyl acetate (clarified lysates5:1; v/v, isolated Δ¹-KstD 2.5:1; v/v, Hep3B and U-937 cells 2:1; v/v).Samples were subjected to centrifugation between extractions at 3,100×gfor 1 minute at 25° C. in order to minimize the interphase layer andimprove extraction efficiency. The organic phase was collected andevaporated under nitrogen gas. Isolated pregnane analytes werereconstituted in an 80:20 mixture of 30% [vol/vol]acetonitrile in H₂Oand 80% [vol/vol] 2-propanol in H₂O. Isolated cholestane analytes werereconstituted in 90% [vol/vol] acetonitrile in H₂O. Clarified bacteriallysates and eukaryotic samples were resuspended in 250 mL and 500 mL,respectively, with the appropriate HPLC sample buffer. Samples werefiltered with Millipore Ultrafree PVDF centrifugal filters (0.1 μm), and80 μL of the sample was injected and analyzed by RP-HPLC.

Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) Analysis

For separation and identification of steroid bioconversion analytes, ananalytical RP-C18 column (Chromolith 100; end capped; 5 m; 100 by 4.6mm; Merck, Darmstadt, Germany) was used with a Hitachi Elite LaChromHPLC equipped with an in-line Perkin Elmer Radiomatic 150TR flowscintillation analyzer. For separation of pregnane based analytes, themobile phase was comprised of a mixture of solvent A (30% [vol/vol]acetonitrile in H₂O) and solvent B (80% [vol/vol] 2-propanol in H₂O).Separation was performed at a flow rate of 0.8 ml min⁻¹ at roomtemperature with a linear gradient starting from 80:20 to 50:50 solventA:B over 30 minutes. For separation of cholestane based analytes, themobile phase was comprised of a mixture of solvent A (90% [vol/vol]acetonitrile in H₂O) and solvent B (85% [vol/vol] acetonitrile in2-propanol). Separation was performed at a flow rate of 1.25 ml min⁻¹ atroom temperature with an isocratic elution of 100:0 solvent A:B fromtime 0-25 minutes, a linear gradient of 100:0 to 0:100 solvent A:B from25-35 minutes, and an isocratic elution of 0:100 solvent A:B from 35-45minutes.

We claim:
 1. A nucleic acid composition comprising: a coding sequence, coding for one or more proteins; and a control sequence, for regulating the transcription of one or more of the coding sequences, wherein the proteins are sterol metabolism proteins.
 2. The nucleic acid composition of claim 1, wherein the sterol metabolism proteins are selected from cholesterol dehydrogenase (CholD), 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), and the coding sequence or amino acid sequence is modified to aid in expressing the proteins in a eukaryotic cell.
 3. The nucleic acid composition of claim 2, wherein the sterol metabolism proteins are humanized forms of 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), and wherein the control sequence is a eukaryotic transcription promoter sequence.
 4. The nucleic acid composition of claim 3, wherein the eukaryotic transcription promoter sequence is a CMV promoter sequence.
 5. The nucleic acid composition of claim 4, wherein the coding sequence comprises the sequence SEQ ID NO:
 27. 6. A method of regulating a sterol concentration in a subject in need thereof, the method comprising the steps of: modifying an immune cell of the subject by introducing a nucleic acid; allowing the immune cell to express one or more sequences from the nucleic acid, wherein the nucleic acid codes for one or more proteins involved in sterol metabolism; administering the modified cell to the subject, and allowing the modified cell to degrade the sterol.
 7. The method of claim 6, wherein the protein is selected from cholesterol dehydrogenase (CholD), 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
 8. The method of claim 7, wherein the one or more sequences code for humanized proteins 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 303-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx) and wherein expression is controlled by a eukaryotic promoter sequence.
 9. The method of claim 8, wherein humanized 3-ketosteroid 9α-hydroxylase (KshAB) is targeted to a mitochondrion of the cell by including a mitochondrial targeting sequence in the KshAB coding sequence.
 10. The method of claim 9, wherein the eukaryotic promoter sequence is a CMV promoter sequence.
 11. The method of claim 10, wherein the nucleic acid has the sequence SEQ ID NO:
 27. 12. The method of claim 11, wherein the immune cell is a monocyte or macrophage.
 13. A method of altering a eukaryotic cell that cannot catabolize a sterol comprising: introducing an expression vector comprising a nucleic acid into the cell; and expressing one or more enzymes that catabolize the sterol from the vector.
 14. The method of claim 13, wherein the sterol is cholesterol and the one or more enzymes are selected from cholesterol dehydrogenase (CholD), 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), anoxic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).
 15. The method of claim 14, wherein the one or more enzymes are 3-ketosteroid Δ¹-dehydrogenase (Δ¹-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx) and wherein expression is controlled by a eukaryotic promoter sequence.
 16. The method of claim 15, wherein humanized 3-ketosteroid 9α-hydroxylase (KshAB) is targeted to a mitochondrion of the cell by including a mitochondrial targeting sequence in the KshAB coding sequence.
 17. The method of claim 15, wherein the eukaryotic promoter sequence a CMV promoter sequence.
 18. The method of claim 16, wherein the eukaryotic cell is an immune cell.
 19. The method of claim 13, wherein the nucleic acid comprises at least one sequence selected from SEQ ID NOS: 10-27.
 20. The method of claim 16, wherein the nucleic acid has a sequence SEQ ID NO:
 27. 